NBDE Part I Biochem Review and Study Guide

2003 Biochemistry Review for the National Boards Part I ©2003 Gene C. Lavers, Ph.D.

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Examination Specifications (2002)1 Building Blocks of Life Course (41) I. Physical-Chemical Principles (3) A. Basic principles (2) B. Applied principles (1) II. Biological Compounds (9) A. Sugars and carbohydrates (1) B. Amino Acids and proteins (2 ) C. Lipids (1) D. Nucleic Acids and metabolism (1) E. Interdisciplinary clinical/cross correlation (4) III. Metabolism (13) A. Bioenergetics (1) B. Enzymology (1) C. Catabolism (2) D. Anabolism (2) E. Regulation (2) F. Interdisciplinary clinical/cross correlation (5)

---25 --IV. Molecular Biology (8) A. DNA/RNA and protein synthesis (4 ) B. Genetic engineering (2) C. Interdisciplinary clinical/cross correlation (2) XIV. Nutrition (8) A. Nutrients/minerals (3) 1. Requirements (1) 2. Functions (2) B. Interdisciplinary and clinical/cross correlation (2)

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---9 of 25 --Biochemistry in other D1 Courses (13) V. Connective Tissues (8) (in Basic Tissues) A. Soft tissue (2) B. Hard Tissue/calcification (3) C. Interdisciplinary and clinical/cross correlation (3) VI. Membranes (4) (most in Cell Organelles ) A. Structure (1) B. Function (1) C. Interdisciplinary clinical/cross correlation (2) VII. Nervous System (9) (most in Organ Systems) A. General properties (2) B. Central nervous system (1) C. Autonomic nervous system (1) D. Somatic nervous system (with reflexes) (2) E. Special senses (1) F. Interdisciplinary and clinical/cross correlation (2) VIII. Muscle (6) () A. Skeletal (2) metabolism and myoglobin B. Smooth (1) C. Cardiac (1) D. Interdisciplinary and clinical/cross correlation (3) IX. Circulation (9) (in Basic Tissues/clotting/gas transport A. Fluid content and dynamics (2) B. Coagulation (1) C. Cardiodynamics and electrophysiology (2) C. Regulation (1) D. Interdisciplinary and clinical/cross correlation (3) X. Respiration (6) (in Basic Tissues) A. Mechanical aspects (1) B. Gas exchange and transport (1) C. Regulation (1) D. Interdisciplinary and clinical/cross correlation (3) XI. Renal (6) (see Organ Systems) A. Functional anatomy (1) B. Blood flow and filtration (1) C. Reabsorption and secretion (1) metabolites D. Interdisciplinary and clinical/cross correlation (2) XII. Acid-Base Balance (1) (in Organ Systems) XIII. Digestion (5) (in Organ Systems) A. Neuromuscular (1) B. Secretions (1) enzymes C. Absorption (1) D. Regulation (1) E. Interdisciplinary and clinical/cross correlation (1) XV. Endocrines (8) (in Organ Systems) A. Pituitary/hypothalamus (1) B. Reproduction (1) C. Signaling systems (2) insulin/glucagon & cAMP D. Pancreas/parathyroid (1) E. Adrenal/thyroid (1) F. Interdisciplinary and clinical/cross correlation (2)


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Integrated Biochemistry Review and Study Questions To assist you in reviewing Biochemistry for the Part I National Boards, review questions were prepared and are accompanied by suggested study or thought questions in red text. Additionally, written answers - as endnotes - are provided to foster easier review rather than new learning. To see the answer, move the mouse cursor over the endnote number (exponent) and the endnote pops up. Greek letters, super/subscripts formatting are lost in the popup note, so consult the actual endnote as needed. [Additional information is within brackets in grey tone, which is also lost in the popup note.]. The TOPIC headings (centered) and their SUBTOPIC headings (left margin) are also in gray scale. Topics in Building Blocks of Life course are in blue text. Other topics taught in the previous traditional biochemistry course are in other D1 courses, these other topics are in green text. Many subtopics can not be included in a short 50 question format, so the related study questions attempt to expand the subtopics in the questions. Not all answers (*) are reviewed or discussed in the endnote material. PHYSICAL-CHEMICAL PRINCIPLES (3) BASIC PRINCIPLES 1. Which of the following functional groups behaves as a weak acid, i.e., dissociate a proton in aqueous solution, at physiological pH? A. R—CH2—R' B. RCH2—OH C. R—NH3+ * D. R—COOH E. Ph—OH Related study questions. [1] What is physiological pH?2 What is a weak acid compared to a strong acid?3 [2] Identify each functional group above, which can dissociate at physiological pH; and why4? [3] Why is the pKa of –COOH and –NH2 on the α-carbon of amino acids about 1-2 pH unit higher and lower, respectively5? 2. Which of the following pairs of compounds is INCORRECTLY matched with its respective type of stereoisomerism? A. D- and L–glyceraldehyde : absolute configuration B. α– and β-glucose : anomer pair C. glucose and mannose : epimers D. cis- and trans–fumarate : geometric isomers * E. ribose and glucose : diastereomers Related study questions. [1] How many configurations can an asymmetric tetrahedral carbon and a chiral carbon have 6? How many different configurations do all of the asymmetric carbons (C*) contribute in a sugar? 7 How many C* in aldoses8; ribose, glucose, and mannose; in ketoses9: ribulose, xylulose, fructose? [2] Regarding carbons C1–C5 or C1–C6, which chiral carbons in those sugars define pairs of anomers (α/β)10, epimers11 (e.g., glucose and mannose) , D/L-sugar families12 (e.g., D/L-glucose). [3] How many of the asymmetric carbon atoms of these sugars rotate polarized light, i.e., are optically active? 13 If a D-sugar always give a (+) optical rotation does the corresponding mirror image L-sugar always give a (–) rotation?14 Is there any consistent relationship between the asymmetrical tetrahedral carbons and the direction (+ or –) of optical light rotation properties?15 [4] What are meso stereoisomers versus diastereoisomer? 16 Cite an example17. APPLIED PRINCIPLES 3. Which thermodynamic parameter deals with randomness and disorder? A. enthalpy * B. entropy C. free energy D. activation energy E. potential energy Related study questions. [1] What is the meaning of each the terms listed18? [2] What is the significance of random disorder versus specified order in biological systems 19?


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[3] Is entropy increased or decreased by such processes as glycogenesis, fatty acid synthesis, protein synthesis, DNA and RNA synthesis20 compared to others (e.g.: glycogenolysis, β-oxidation, proteolysis, and hydrolysis of DNA and RNA)21? BIOLOGICAL COMPOUNDS (9) SUGARS AND CARBOHYDRATES 4. The C1 of ribose and glucose has all of the following properties EXCEPT ONE. Which is the EXCEPTION? A. Anomeric carbon forms α/β racemic mixture. B. Acyl aldehyde groups react with primary and secondary alcohols, amines, and mercaptans. * C. D/L isomers define the specific optical rotation. D. In solution, mutarotation of either pure intramolecular hemiacetal yields an equilibrium mixture of α/β anomers. E. Forms acetal ribosides and glucosides, respectively, that can’t mutarotate. Related study questions [1] How do planar aldehydes and ketones groups give rise to an asymmetric tetrahedral carbon racemic pair 22? [2] Compare the total number of asymmetric centers in the open-chain and cyclic-forms of aldoses ribose, deoxyribose, glucose, mannose, galactose have (and ketose fructose) 23. [3] Mutarotation may or may not invert C1 configuration of cyclic-aldoses, but when C1 is in an α(1-4) acetal linkage no inversion is possible, for example, for the C1 of the α−glucosyl moiety of maltose (glucosyl−α(1-4)-glucose), or for the β−glucosyl moiety of cellobiose (glucosyl−β(1-4)-glucose) or for the β−ribosyl moiety of adenosine (N−β−ribosyl adenine); explain.24 [4] Does this acetal type of chemistry have significance for RNA and DNA stability and function? 25 AMINO ACIDS AND PROTEINS 5. All of the following are characteristic of amino acids EXCEPT. A. The α-carbon (C2) is a chiral center (except for glycine). * B. All migrate to the cathode in an electric field at physiological pH. C. They have side chains with different physical and chemical properties. D. Most can form dipolar ions (zwitterions) at physiological pH. E. Only a repertoire of 20 amino acids are incorporated into proteins. [1] How do D- and L-amino acids relate to the D- and L-glyceraldehyde reference compounds?26 [2] What functional groups are present in the side-chains of free amino acids that account for their ionization to zwitterion forms at physiological pH?27 [3] How do some amino acids acquire a net (+1) or (–) charge in aqueous solution?28 [4] What changes in net charge occur in free amino acids if the pH is adjusted from pH 2 to pH 12 that effects their net charge and therefore their electrophoretic mobility at different pHs?29 [5] Which amino acids are always incorporated into proteins compared to others in the body that arise either as pathway metabolites (e.g., ornithine in the urea cycle) or as a result of posttranslational modification of in a protein (e.g., hydroxyproline from proline hydroxylation in collagen chains? 30 6. Which amino acid is INCORRECTLY matched to its side-chain? A. lysine : ε−amino–aliphatic hydrocarbon chain B. isoleucine : branched aliphatic hydrocarbon chain * C. tyrosine : aromatic imidazole D. glutamic acid : δ–carboxylate –aliphatic hydrocarbon chain E. methionine : γ-methylmercapto–aliphatic hydrocarbon chain Related study questions [1] How is the tertiary (3°) and quaternary (4°) structure of proteins affected by the properties of the amino acid side-chains (polar, apolar, acidic, basic, neutral, aliphatic, aromatic, and reactivity)? 31 [2] Which amino acids are usually in the hydrophobic interior and which are on the hydrophilic exterior in contact with water when the primary sequence folds to yield the functional 3D-structure? 32 [3] Which amino acids are most often modified with sugars and lipids posttranslationally that are then involved in non-bonded versus covalent cross-links in complex structures? 33 LIPIDS 7. Select the INCORRECT statement about lipids.


*

A. B. C. D. E.

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Lipids are small nonpolar molecules extracted by organic solvents. Most of our fat is stored as triglyceride droplets in adipose tissue cells. Phospholipids are the major class of lipids in membranes. ABO-blood group antigens are glycosphingolipids (glycolipids). Most fatty acids are present as free acids in cells.

Related study questions [1] What are the major classes of lipids and their constituent compounds found in the body? 34 [2] What are some of the key properties of lipids that make them suitable for energy storage, as thermal and electrical insulators, and appropriate for membrane partitions?35 [3] What core component distinguishes phospholipids from sphingolipids 36; what carbohydrate components of glycosphingolipids constitute the ABO blood group antigens on red blood cells? 37 NUCLEIC ACIDS AND METABOLISM 8. Which one of the following compounds DOES NOT supply atoms for the synthesis of the purine ring system? A. glycine B. aspartate C. N10-formyl-THFA D. CO2 * E. S-adenosyl methionine (SAM) Related study questions [1] What are the substrates and sequence of incorporation of atoms or fragments into the purine ring system during de novo synthesis of IMP?38 [2] How is IMP converted to AMP or GMP?39 Why are some purine and pyrimidine reaction steps coupled to ATP hydrolysis? 40 [3] Choices C to E are compounds involved in 1-carbon fragment metabolism for the synthesis of which nucleotides? 41 INTERDISCIPLINARY CLINICAL/CROSS CORRELATIONS 9. All of the following EXCEPT one correctly completes the statement below. Which is the EXCEPTION? Oxidation of glutathione (GSH) to disulfide GSSG in red blood cells protects proteins from oxidative damage by: A. O2•–. B. reactive oxygen species. * C. NADP+. D. methylglyoxal. Related study questions [1] What is glutathione (GSH), which of its functional groups has an antioxidant role against various reactive oxygen species (ROS), and which two cells-types have GSH levels that reach 6-8 mM?42 [2] The glyoxalase pathway in all cells protects proteins against oxidative insult from what small glucose fragments originating from glycolysis?43 [3] What is the chemical relationship of oxygen, oxygen superoxide, hemoglobin, methemoglobin, and Heinz bodies? 44 10. Gout disease and Lesch-Nyhan syndrome are due to deficiency of one or two enzymes in purine metabolism and can be treated with allopurinol. Which statement about allopurinol biochemistry is INCORRECT? A. Allopurinol decreases the rate of oxidation of purines to urate. * B. Allopurinol increases urate solubility. C. Allopurinol inhibits xanthine oxidase. D. Allopurinol acts as a suicide inhibitor. E. Allopurinol increases the opportunity for purine salvage. Related study questions [1] What two salvage enzymes and their reactions are deficient in purine metabolism that leads to hyperuricemia and excessive accumulation of poorly soluble urate in the joints of cooler extremities and kidney stones in middle-aged men? 45 [2] What molybdenum-requiring enzyme is inhibited by allopurinol? Why is allopurinol an example of a suicide inhibitor? 46 [3] Account for the effect of allopurinol in regulating purine biosynthesis and alleviating symptoms? 47 11. Which of the following represents the chemical substance that is an additional reserve of energy for muscle contraction? A. Glycogen B. Lactic acid

2003 Biochemistry Review for the National Boards Part I ©2003 Gene C. Lavers, Ph.D. *

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C. Acetyl CoA D. Creatine phosphate E. Adenosine triphosphate

Related study questions [1] What is the primary source of energy for muscle contraction? 48 [2] What compound is the secondary backup energy reserve the muscle cell used to regenerate ATP, which is rapidly depleted during short bursts of contraction thereby giving time for activation of glycogenolysis to supply glucose thereafter? 49 [3] Why is creatine phosphate a high energy molecule like ATP?50 [4] What dead-end catabolite of creatine phosphate is excreted from the muscle cell into plasma and then into urine that betrays bursts of vigorous muscle contraction?51 12. A severe pyruvate carboxylase (PCase) deficiency would likely produce TCA cycle dysfunction and very low ATP levels in cells because: A. pyruvate concentration is too low. B. lactic acid concentration is too low. C. of insufficient fatty acid synthesis for membrane formation. D. of respiratory acidosis from ongoing rapid breathing. * E. of insufficient oxaloacetic acid (OAA) to condense with acetyl CoA to form citrate. Related study questions [1] What are anaplerotic reactions and why is pyruvate carboxylase so critical for normal TCA cycle activity? 52 [2] Why do high pyruvate and lactate levels occur in PCase deficiency; why would that lead to lactic acidosis? 53 [3] Why does lipogenesis increase significantly (up to several fold during this deficiency); 54 and what pathway maintains the supply of NADPH required?55 METABOLISM (13) BIOENERGETICS 13. Hexokinase __1__ the free energy from ATP hydrolysis to phosphorylate glucose thereby driving an unfavorable endergonic reaction with an spontaneous __2__ reaction. A. 1 = uncouples 2 = endergonic B. 1 = uncouples 2 = exergonic C. 1 = couples 2 = endergonic * D. 1 = couples 2 = exergonic Helpful hints [1] What is the significance of free energy change (∆G) compared to free energy (G); what is enthalpy change, in biological systems and why is ∆G more useful as a measure of metabolic achievement? 56 [2] If ∆G°´ = – 7.3 kcal/mol for ATP hydrolysis (Rx I): ATP + H2O –> ADP + Pi; and if ∆G°´ = + 3.3 kcal/mol for the phosphorylation of glucose using a phosphate (Rx II): glucose + P –> G-6-P; then account for the – 3 kcal/mol net ∆G°´ that results from the coupled reaction (Rx III) of hexokinase: glucose + ATP –> G-6-P + ADP?57 [3] Reactions that are spontaneous are generally exergonic. In the individual and coupled reactions in the review question [2], which reaction is exergonic, which is endergonic?58 Overall, is catabolism exergonic or endergonic; is anabolism exergonic or endergonic?59 ENZYMOLOGY 14. Which term of the Michaelis-Menten equation,

v=

*

Vmax [ S ] Km + [S]

can be determined from the x-intercept of a Lineweaver-Burke plot? A. v B. Vmax C. Km D. [S]

Related study questions [1] What is a double reciprocal plot, and why is it advantageous in determining K m and Vmax?60 [2] What is the meaning of Km and Vmax for an enzyme reaction?61


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[3] Why is the initial velocity (v o) important for determining v of an enzyme reaction; what are zero and first order kinetics, relative the Km?62 CATABOLISM 15. All the following reactions occur during glycolysis EXCEPT ONE. Which is reaction is the EXCEPTION? A. fructose-6P + ATP (phosphofructokinase–1)  fructose-1,6-BP * B. fructose-2,6-BP (aldolase)  2 glyceraldehyde-3P C. glyceraldehyde-3P + NAD+ (G3P dehydrogenase)  1,3-bisphosphoglycerate + NADH D. 1,3-bisphosphoglycerate + ADP (phosphoglycerate kinase, PK)  3-phosphoglycerate + ATP E. pyruvate + NADH (lactate dehydrogenase)  lactate + NAD+ Related study questions and perspectives on glycolysis [1] What is the purpose of glycolysis63; why is reaction ‘A’ the committed step?64 What is the major product of aerobic versus anaerobic glycolysis?65 [2] The red blood cell (RBC,) like all cells, contains only trace quantities of coenzymes (hence “catalytic quantities”). The RBC would convert the tiny supply of NAD+ to NADH very quickly and then glycolysis would stop! Which listed enzyme reaction ‘?’ prevents this metabolic collapse? 66 [3] Reactions C and D constitute a catalytic process known as substrate level oxidative phosphorylation, which is one of two substrate level means of ATP regeneration (from ADP) essential for sustaining all ATP-dependent RBC metabolic reactions from collapse. Ironically complete reliance on C and D produces a lower net ATP yield compared to all other cells. What cellular organelle is uniquely missing in the RBC that other cells have and rely upon extensively for most ATP regeneration and thereby bypass cytosolic lactate dehydrogenase to regenerate their NAD+?67 [4] Which highly exergonic enzyme reaction (not listed, which in contrast to D doesn’t need an oxidative reaction like C), provides the second substrate level phosphorylation of ADP in the RBC (all other glycolytic cells too)? 68 [5] In other cells, which have mitochondria and can funnel NADH into both cytosolic lactate dehydrogenase or the mitochondrial respiratory chain (in proportion to dynamic hypoxic oxygen levels), the net ATP regeneration ratio goes up or down between the extremes of 2 and 8 ATP per glucose oxidized. What is the name given to glycolysis at either extreme?69 [6] With the above in mind, can you appreciate why the glycolytic throughput must end in lactate not pyruvate? Hence, the major source of plasma lactate is understood? 70 What other major tissue can produce and secrete massive quantities of lactate into the plasma using glycolysis? 71 16. Which of the following glycolytic liver enzymes is NOT product-inhibited and is absent in muscle cells? A. Hexokinase B. Enolase C. Aldolase * D. Glucokinase E. Glucose-6-phosphatase Related study questions [1] After a meal, glucose uptake in liver cells is facilitated and rapid compared to muscle cells because liver has an additional glucose kinase that is not product-inhibited and has a higher Km for glucose. Why are these distinctions important for appreciating special liver cell functions compared to muscle cell function?72 [2] An abrupt and sustained increase in diet glucose induces gene expression of one of these liver kinases, which enzyme is inducible and why is that advantageous for liver functions?73 ANABOLISM 17. Select the statement about glycogen biochemistry that is INCORRECT. A. Glycogen is composed of highly branched chains of α−glucose residues. B. Glycogen’s outward branching provides reactive ends for extremely rapid incorporation and release of glucose. C. Glucose residues are linked by acetal α(1  4) bonds. D. Approximately 1 in 8 glucose residues are linked by an acetal α(1  6) bond. * E. Glycogen is hydrophilic and therefore is less dense than triacylglycerides. Related study questions [1] What special aspects of acetal linkage properties and associated enzymes are advantageous with respect to storage of glucose in starch and glycogen or for use of glucose for structural strength characteristic of cellulose? 74


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[2] Explain how the highly branched polymer structure of glycogen provides a major advantage compared to the structure of the linear starch polymer in facilitating different in rates of synthesis and breakdown (i.e., storage and release) for both polymers?75 [3] Is this consistent with muscle using glycogen rather than starch, which is used in plants? 76 18. Unlike β-oxidation of fatty acids, fatty acid synthesis requires only: A. NAD+ * B. NADPH C. FAD D. FADH2 Related study questions [1] In the fatty acid synthesis pathway, which two reactions use NADPH; in contrast, which of the listed redox coenzymes are used in the β-oxidation catabolic pathway? 77 [2] Which metabolic pathway(s) or reaction(s) supply NADPH for FA synthesis; which vitamin, if deficient, therefore interferes with FA synthesis, which second vitamin, if deficient with the first interferes with both FA pathways? 78 [3] Which inherited enzyme deficiency was first revealed when an antimalarial (primaquine) was prescribed that lead to insufficient NADPH production in the hexose monophosphate shunt in RBCs that developed into hemolytic anemia? 79 REGULATION 19. Regulation of glycolytic enzyme activity includes posttranscriptional phosphorylation/dephosphorylation of: * A. pyruvate kinase B. glycogen synthase C. phosphorylase kinase D. phosphorylase a E. phosphofructokinase-1 Related study questions [1] A variety of regulatory mechanisms control the flux of molecules through the glycolytic pathway. These include substrate availability, inhibition, allosterism, and posttranslational modification. Which enzymes are regulated by these mechanisms? 80 [2] Which small glycolytic molecules, including those from other pathways, are involved in the regulatory mechanisms of glycolysis?81 [3] Which pancreatic hormone(s) stimulates or inhibits glycolytic activity? 82 By what mechanisms?83 20. Which key substrate of fatty acid synthesis also controls the inhibition of β-oxidation and thereby prevents a futile cycle? A. acetyl CoA * B. malonyl CoA C. citrate D. pyruvate E. propionyl CoA Related study questions [1] What is a futile cycle; why would metabolic futile cycles be life threatening? 84 [2] What is the activated substrate for cytosolic fatty acid synthesis; which molecule inhibits the enzyme-driven entry of acyl CoA fatty acids into the mitochondria for β-oxidation?85 How do those properties prevent a futile cycle in fatty acid metabolism?86 [3] Excess dietary glucose is easily converted to fat and stored. Oddly enough, acetyl CoA arises from pyruvate via cytoplasmic glycolysis of the excess glucose. Why does acetyl CoA become trapped inside the mitochondrion after mitochondrial pyruvate dehydrogenase acts on pyruvate? 87 [4] How does acetyl CoA escape the mitochondrion88; how does acetyl CoA (and OAA) arise from cytosolic citrate89; how is acetyl CoA activated with CO2 to form malonyl CoA.90 [5] How does propionyl CoA arise from β-oxidation of odd-numbered fatty acids?91 INTERDISCIPLINARY CLINICAL/CROSS CORRELATION 21. Nucleoside derivatives are effective antibiotics compared to their free bases because after phosphorylation to nucleoside triphosphates, the appropriate cellular polymerases then incorporate them into A. the primary sequence of full length histone nucleoproteins in bacteria interfering with transfection.


*

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B. polysaccharides that unfold DNA supercoils interfering with chromosome replication. C. the 5'terminal cap of hnRNA pre-mRNA interfering with posttranscriptional processing. D. RNA or DNA transcripts either for subsequent inhibitory effects or termination of further polymerization.

Related study questions [1] What is azido thymidine, dideoxyadenosine, and puromycin; what structural features make each of these nucleosides effective antibiotics due to their mode of action?92 [2] Which cellular enzymes interconvert nucleosides and nucleotides, exchange sugars of nucleosides, salvage purine and pyrimidine bases directly to nucleotides thereby allowing nucleotides and their drug analogs metabolic effectiveness? 93 [3] Cite at least one example of a nucleoside with a modified sugar that terminates DNA synthesis thereby inhibiting productive infection by the AIDS virus?94 22. An immigrant migrant farm worker presents her 5-year old male child to the emergency room complaining “All his teeth are rotting, he cries all the time and seems to be in pain.” Examination reveals that the boy is unable to understand and answer simple questions and has an unusual gait, sitting posture, and very light pigmentation. The family history indicates male mental retardation and epilepsy, but otherwise normal dental histories. Urine analysis indicates excessive phenylpyruvate, phenyllactate and a mousey smell. Further tests confirm phenylalanine hydroxylase deficiency. The most likely diagnosis is: A. Albinism. B. Alkaptonuria. C. Maple syrup urine disease (MSUD). * D. Phenylketonuria. Related study questions [1] What simple test detects phenylketonuria (PKU) shortly after childbirth because it is mandated by law in most states, but often is not performed in foreign countries? Which aspect of metabolism is affected? 95 [2] What adjustment is made to the dietary formula to prevent mental retardation from developing in the infant? 96 [3] Explain why a patient with PKU is on the horns of a dilemma in trying to balance a possible essential amino acid deficiency against neurotoxicity of phenylalanine?97 [4] Why is rampant caries and very light pigmentation both irrelevant for the diagnosis of this case? 98 [5] Why can a tyrosine supplement compensate for occasional unintended phenylalanine dietary excess? 99 23. Jaundice in a patient with a history of alcoholic cirrhosis is most likely due to a metabolic dysfunction in: A. bilirubin synthesis by the liver. * B. bilirubin catabolism by the liver. C. cytochrome synthesis by the pancreas. D. cytochrome catabolism by the pancreas. Related study questions [1] What is the pathway of heme synthesis and catabolism?100 [2] Where do each of these synthetic and catabolic reactions occur? 101 [3] What are jaundice and hepatomegaly? 102 24. All of the following correctly complete the sentence about sickle-cell anemia EXCEPT ONE. Which is the EXCEPTION? “Sickle cell anemia was the first complex disease discovered to be due to … A. a single nucleotide mutation in the gene coding for β-hemoglobin A.” * B. a nonsense codon mutation in the gene coding for β-hemoglobin A.” C. an altered hemoglobin conformation that promotes insoluble fibril formation.” D. distorted cell shape and cell inflexibility that leads to blocked capillaries and localized micro hypoxia. ” E. a variety of clinical signs and symptoms. ” Related study questions [1] What is the nature of the mutation in sickle cell anemia? 103 [2] What causes the change in shape and flexibility during hypoxia? 104 [3] Why does sickle cell anemia show a variety of clinical signs and symptoms? 105 25. All of the following are true statements concerning zymogen activation cascades EXCEPT ONE. Which is the EXCEPTION? Zymogens: A. are often triggered in emergency situations when rapid response is essential. B. provide very rapid amplification of active enzyme catalysis. C. avoid life-threatening circumstances by accelerating enzymological responses.

2003 Biochemistry Review for the National Boards Part I ©2003 Gene C. Lavers, Ph.D. *

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D. provide a mechanism for rapid serial feedforward catalytic activations within a response pathway. E. provide critical cCMP hydrolysis activations for rapid signal transduction mechanisms.

Related study questions [1] What is a zymogen?106 [2] Why do we need zymogen cascades?107 [3] Which body systems require zymogen cascades? 108 MOLECULAR BIOLOGY (8) DNA/RNA AND PROTEIN SYNTHESIS 26. All of the listed proteins EXCEPT ONE contains an N-terminal signal peptide that is bound by the Signal Recognition Particle (SRP) and is subsequently cleaved as a posttranslational modification. Which nascent protein lacks the signal peptide and is not exported by the cell? A. Collagen B. Fibrinogen * C. Tubulin D. Immunoglobin E. Fibronectin Related study questions [1] What is unique about the amino acid sequence of the signal peptide and where in the polypeptide chain is it located? 109 [2] How does the signal peptide differentiate ribosomes synthesizing collagen, fibrinogen, plasma fibronectin, and immunoglobin polypeptide chains for excretion from other ribosomes synthesizing proteins that will remain inside a fibroblast, liver, or immune cell?110 Define intracellular versus an extracellular protein, and endogenous versus exogenous proteins from those listed?111 [3] What is the cytoplasmic SRP complex that sorts proteins for secretion or retention based upon the signal peptide? 112 [4] Why do ribosomes dock to the endoplasmic reticulum; what is the fate of the signal peptide; what is the fate of the remaining –COOH end of the newly synthesized polypeptide; what organelle(s) may perform additional posttranslational modifications?113 27. The TATA box is important for initiation of which process? A. mRNA capping B. DNA replication and * C. RNA transcription D. RNA translation E. HnRNA splicing Related study questions [1] What is a nucleic acid consensus sequence, where are they located and what type of proteins interact with them? 114 [2] What is the role of the TATA box in a promoter, do all genes have this sequence, if not, cite an example? 115 [3] What consensus sequences are involved in mRNA synthesis, processing, and translation? 116 [4] What role does capping and polyadenylation play in eukaryotic mRNA maturation and translation? 117 28. Which statement is INCORRECT about the typical purine-rich, AGGAGG consensus sequence in bacteria and bacteriophages? A. It is approximately 10 nucleotides upstream from the initiator AUG codon. * B. It is usually capped with m7GpppG. C. It ensures appropriate polycistronic translation. D. It binds near the 3’ terminus of 16S ribosomal RNA. E. It is called the Shine-Dalgarno in mRNA. Related study questions [1] Compare structures of prokaryotic mRNA and eukaryotic mRNA that are involved in the initiation phase of protein synthesis?118 [2] How does the Shine-Dalgarno sequence align the ribosome with the AUG start codon? 119 In polycistronic prokaryotic mRNA, how does the Shine-Dalgarno ensure all cistrons of bacterial mRNA are translated? 120 [3] Why would coordinated multi-protein expression fail in polycistronic mRNA without the purine-rich Shine-Dalgarno sequence?121 29. The m7GpppN cap is a posttranscriptional modification of eukaryotic:


*

A. B. C. D. E.

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DNA introns histones tRNA hnRNA rRNA

Related study questions [1] What aspect of the Central Dogma (Watson and Crick, 1953) does the question address? 122 [2] What is a posttranscriptional modification; which modifications occur during and which after transcription is complete? 123 [3] Which structural modification requires, GTP and S-adenosyl methionine (SAM); which need only SAM?124 [4] Which initiation factor binds recognizes the +1 charge in the 5’-m7GpppN cap of eukaryotic mRNA that facilitates translation?125 [5] Which three metabolic coenzymes have a structural feature similar to the mRNA cap? 126 30. A sample of DNA from a patient’s amniotic fluid cells is prepared for DNA fingerprinting by treatment with an enzyme that will hydrolyze specific phosphodiester bonds of both strands within the sequence, 5’–GAATTC –. Which type of enzyme is used? A. topoisomerase * B. restriction endonuclease C. ligase D. exonuclease E. polymerase Related study questions [1] What substrates do restriction enzymes, topoisomerases, ligases, exo- and endonucleases, and polymerases act on? 127 [2] What role does each listed enzyme perform in a bacterial cell, in a human cell;128 how does the cell distinguish its DNA from foreign DNA in order to abort the biological threat of the foreign DNA? 129 [3] What is a DNA fingerprint; what applications does it have in medicine? 130 GENETIC ENGINEERING 31. By 1975, the cloning of dsDNA fragments became possible after which pair of enzymes were discovered? * A. DNA restriction enzyme and DNA ligase B. DNA restriction enzyme and polynucleotide phosphorylase C. DNA endonuclease I and DNA ligase D. DNA polymerase I (Kornberg) and DNA ligase E. Topoisomerase I and DNA ligase Related study questions [1] What is the substrate specificity of hydrolytic DNA restriction enzymes; why must a cell methylate its DNA if it expresses a restriction enzyme?131 What are blunt ends compared to cohesive or “sticky” ends produced by restriction enzymes? 132 [2] What does DNA ligation accomplish; why are DNA-ligases NAD- or ATP-dependent? Why do DNA ligases yield stable recombinant DNA molecules suitable for molecular cloning [3] What aspect of the polymerase reaction do DNA ligases mimic, but also lack that prevents them from being a DNA polymerase?133 [4] What is similar/dissimilar about DNA topoisomerases and ligases? 134 [5] Compare RNA polymerase to polynucleotide phosphorylase (PNP) discovered in 1955, in the bacterium A. vinelandii?135 32. Why are plasmids used for molecular cloning of DNA designed with two antibiotic resistance genes? A. Double resistance ensures replication and resistance of host cell to the recombinant. B. Double resistance boosts plasmid copy number and replication efficiency in the host cell. * C. Double verification that recombination occurred and that the host cell was successfully transfected. D. Double verification that recombinant plasmid can kill transfected cells, leaving uninfected cells for cloning. Related study questions [1] What is a plasmid, transfection, plasmid copy number, an antibiotic resistance gene? 136 What is the connection between ampicillin, tetracycline, and plasmids?137 [2] DNA inserted into a gene for drug resistance abolishes the drug resistance for the cell. Does the loss of drug resistance verify that the DNA insertion was successful?138 [3] What is the rationale for putting two antibiotic resistance genes into a plasmid such that one gene contains a restriction site? 139 INTERDISCIPLINARY CLINICAL/CROSS CORRELATION


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33. A newly arrived short, 50-year-old female visiting from a remote region of Central America weighs 250 lbs and presents to an oral surgery clinic. She is referred to the hospital’s diabetes clinic. In spite of daily insulin (porcine) injections for 4 months, her glycosylated HbA1c is 14% (normal 6%), and she has a high titer of antibodies to insulin. The clinic physician will most likely conclude that: A. she must be put on a low carbohydrate diet immediately. B. her carbohydrate metabolism was not controlled for the last 3-months. C. her immune response to porcine insulin has made her hyperglycemic. D. she must be put on cloned human insulin to bypass the immunity to the porcine insulin. * E. all of the above. Related study questions [1] Why is glycosylation of HbA1c of 9-12% indicative of hyperglycemia and poor management of dietary glucose? 140 [2] Explain why diabetes is referred to as “starvation in the presence of plenty.” 141 [3] Explain how type 1 and 2 diabetes mellitus are similar and dissimilar? 142 [4] Why is use of animal insulin for treatment of diabetic patients problematical whereas human insulin is not? 143 [5] Why is molecularly cloned human insulin preferred for treatment of diabetes? 144 34. Which of the following antibiotics would most likely be administered to treat an oral cancer by specifically inhibiting the complex enzyme reaction: dUMP  dTMP? A. Actinomycin D * B. Methotrexate C. Puromycin D. Tetracycline E. Trimethoprim Related study questions [1] What metabolic enzyme or process does each of the listed antibiotics inhibit? 145 [2] What is methotrexate; what other drugs resemble methotrexate and are folate antagonists? Why must the patient be injected with tetrahydrofolate within 15 minutes after administration? 146 [3] What biochemical process(es) do Actinomycin D, puromycin, tetracycline, and trimethoprim inhibit? 147 [4] Which bodily cells would be most affected by methotrexate, in addition to the cancer cells? 148 [5] Which of the listed antibiotics are effective in prokaryotes, which are effective in eukaryotes? 149 NUTRITIION NUTRIENTS/MINERALS 35. Which of the following B-complex vitamins should NOT be administered to correct the respective deficiency disease? A. B1 thiamine : beriberi * B. B2 riboflavin : megaloblastic anemia B. B3 niacin : pellagra C. B6 pyridoxine : neurologic disease D. B12 cobalamin : pernicious anemia Related study questions [1] Of the listed vitamins, which is a coenzyme, which is inactive until combined with other molecule(s)? 150 [2] Which vitamin(s) is involved in oxidation-reduction reactions; in 1-carbon transfers, in 2-carbon transfers? 151 [3] Which are water soluble vitamins, which are fat soluble vitamins? 152 [4] What is at least one major characteristic of each deficiency disease? 153 36. Dietary deficiency of vitamin B6 significantly affects the metabolism of: * A. amino acids by decreasing transamination reactions. B. nucleic acids by increasing synthesis. C. fatty acids by decreasing their activation. D. carbohydrates by increasing glucosamine synthesis. Related study questions [1] Of the three forms: pyridoxine, pyridoxal, and pyridoxamine, which is the major form of B6 in the diet, which is the active form of the vitamin, and which form of B6 or corresponding phosphates are water or lipid soluble?154 [2] Which form is usually involved in a Schiff base intermediate that can yield an amination/deamination, decarboxylation, or dehydration reaction?155


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[3] What enzyme reaction, using B6 as cofactor, is the first step in the synthesis, catabolism, and interconversion of amino acids?156 37. Which of the following metabolic processes/compounds would be negatively affected in copper and iron deficiency anemia? * A. oxidative phosphorylation, oxygen transport, and myoglobin B. glycolysis, glycogenesis, and respiratory cytochromes/nonheme FeS C. lipogenesis, ketogenesis, and transferrin D. oxidative phosphorylation, tricarboxylic acid cycle, and ceruloplasmin Related study questions [1] What trace metal nutrients are essential for respiratory transport chain enzymes, adequate hematocrit for oxygen transport, and myoglobin retention of oxygen in muscle?157 [2] Although a muscle protein for iron storage, the traces of serum ferritin level is 99% specific for which anemia? 158 [3] Which plasma copper-carrying protein scavenges superoxide and other oxygen free radicals that damage plasma proteins (including hemoglobin) and is defective in Wilson’s disease? 159 [4] What effect does copper deficiency anemia have on iron transport, on methemoglobin level and on the synthesis of two important connective tissue proteins; what kinds of anemia are associated with copper and iron deficiencies? 160 REQUIREMENTS 38. Inadequate dietary intake of essential amino acids can lead to metabolic disturbances such as: * A. negative nitrogen balance B. positive nitrogen balance C. excessive protein synthesis D. increased blood volume E. increased lipogenesis and glycogenesis Related study questions [1] Why are eight of the 20 amino acids incorporated into proteins essential or indispensable to the body; identify them? 161 [2] What is nitrogen balance; why does protein metabolism account for most biochemically usable nitrogen in the body? 162 [3] What is the best source of protein for humans, i.e., has optimal amino acid ratios, complete digestibility, absorption, and utilization?163 [4] If dietary methionine suddenly drops to 50% of the minimal recommended daily allowance (RDA), why does protein synthesis in all tissues decrease by approximately the same percentage until methionine intake increases to the RDA? 164 [5] During essential amino acid deficiency, why does the body begin to lose essential nitrogen from its amino acids and proteins?165 FUNCTIONS 39. Two key enzymes, dTMP synthase and deoxyribonucleoside diphosphate reductase, are involved in synthesis of deoxyribonucleotides from their corresponding ribonucleotides. Which coenzyme listed below is neither the reactant nor product form of the coenzymes involved in those reactions? A. N5,10-methylene tetrahydrofolate B. dihydrofolate C. NADPH * D. NADH2 Related study questions [1] In humans, all DNA nucleotides are synthesized from their RNA counterparts via two key enzyme reactions, one reduces ribose to deoxyribose, the other methylates uracil to thymine, outline both reactions. 166 [2] For the conversion of dUMP to dTMP, outline the two essential ancillary reactions that keep it going. 167 [3] Both ancillary reactions utilize coenzymes that are vitamin-dependent. Which vitamins are they? 168 [4] Conversion of ribose to deoxyribose involves a hydride transfer. What is a hydride; which vitamin component of the coenzyme involved transfers the hydride to C2 of ribose?169 [5] dTMP synthesis involves a 1-carbon fragment transfer using folate. What are the two other carriers of 1-carbon fragments found in metabolism, both of which participate in nucleic acid syntheses? 170 40. Which of the following is INCORRECT about zinc? A. Zinc is involved in carbohydrate and energy metabolism. B Zinc is involved in synthesis of metallothioneine, which binds Zn for its absorption in the gut. * C. Zinc is a divalent heavy metal that is as toxic as lead.


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D. Zinc, in increased oral doses, interferes with copper absorption and deficiency. E. Zinc deficiency affects growth, impairs of taste and smell, and delays wound healing. Related study questions [1] Which carbohydrate metabolism enzymes require zinc, i.e., their holoenzyme is an active zinc metalloenzyme, but their nonmetalloenzyme apoenzyme is inactive?171 [2] What is metallothioneine protein, where is it synthesized, and what is its role in trace metal absorption? 172 [3] What is the connection of Wilson’s disease to copper and zinc metabolism and likely effect on enzymes requiring them? 173 INTERDISCIPLINARY AND CLINICAL/CROSS CORRELATION 41. Which vitamin is INCORRECTLY paired with the consequence(s) of its deficiency? A. vitamin D : rickets B. vitamin C : scurvy C. niacin : dermatitis, diarrhea and dementia * D. thiamine : polycythemia E. folate : anemia Related study questions [1] Of the vitamins listed, which tissue(s) is usually affected; what signs and symptoms are observed by clinicians during examination of patients with the listed deficiency diseases; what deficiency disease corrects the INCORRECT answer pair?174 [2] Which of the vitamins listed are water soluble; which are lipid soluble? 175 [3] What foods are usually recommended to restore health, if that is possible? 176 42. A dentist recommends postponement of oral surgery upon learning the patient has leukemia and is scheduled for folate antagonist (methotrexate) treatment in two days. The postponement recognizes the complications of antifolate biochemistry and its effects on post operative tissue healing. Which is INCORRECT about folate antagonists? They… A. indirectly block the synthesis of dTMP from dUMP by thymidylate synthase. B. inhibit dihydrofolate reductase. C. mimic the isoalloxazine ring of tetrahydrofolate. * D. block hydride transfer by nucleoside diphosphate reductase. E. treat blood leukemia disorders. Related study questions [1] Why does methotrexate indirectly inhibit the thymidylate synthase and also indirectly inhibit all other tetrahydrofolatedependent metabolism?177 [2] What glucose pathway supplies NADPH required to regenerate dihydrofolate (FH 2) to tetrahydrofolate (FH4)?178 [3] A part of DNA synthesis is serine-dependent. What part of serine’s structure supplies the 1-carbon fragment subsequently added to N5 in the isoalloxazine ring of folate?179 [4] N5,N10-methylene tetrahydrofolate is converted to what folate form by thymidylate synthase that has no other metabolic utility, i.e., is a dead-end metabolite?180

Biochemistry in other D1 Courses (13) CONNECTIVE TISSUES SOFT TISSUE 43. Identify the INCORRECT statement about collagen: “Collagen is an important component of extracellular matrix (EMC) in connective tissues because the … ” A. repeated Gly-X-Y tripeptide in all three subunit chains permits the required tertiary and quaternary structure for collagen’s structural role.” B. tertiary structure of each of the triple strands coils into a left-handed, α-helical conformation.” C. triple-stranded, cross-linked, right-handed super helical quaternary structure forms strong, stable, rigid, rod-like molecules that aggregate into fibrils.” D. hydroxyproline on the surface can be linked to various carbohydrates. “ * E. triple stranded subunits are also stabilized by a cluster of cystine cross links.” Related study questions 8888 Endnotes editing stops here 8888 [1] What is extracellular matrix; why is it needed in connective tissue? 181 [2] What are the chemical features of collagen’s repeated tripeptide Gly-X-Y that are responsible for collagen’s unique 2º, 3º, 4º structures and role in connective tissue?182


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[3] What intermolecular arrangement accounts for the banded appearance of fibrillar collagen? 183 HARD TISSUE 44. Hyaluronic acid, heparin, and chondroitin-, dermatan-, heparin-, and keratin-sulfates are found in: A. anticoagulants * B. proteoglycans C. hormones D. fibronectins E. simple lipoproteins Related study questions [1] What are simple- and derived-sugars; which are found in complex carbohydrates? Which sugar is in RNA versus DNA? 184 [2] What are the different chemical forms of glycoconjugates; which are classified as glycolipids? 185 [3] Which functional groups are added to glucose, galactose, mannose, glucuronic acid, etc., that give rise to the carbohydrates and their molecular categories listed in the question.186 45. Which property about enamel is INCORRECT in comparison to other mineralized tissues within the body? A. Enamel is the hardest. B. Enamel contains mostly hydroxyapatite. * C. Enamel contains amelogenin proteins. D. Enamel contains hexagonal rod crystals that are larger and more firmly packed. Related study questions [1] What are the seven systems that all crystalline minerals are categorized into; calcium phosphate (empirical formula: Ca10(PO4)6(OH)2) a member of which system? What is its common mineral name?187 [2] Is this mineral harder than calcium phosphate mineral found in bone? What scale is used to determine/classify mineral hardness?188 [3] What are amelogenins and where are they found in hard tissue? What features make them suited for their role in mineralization?189 [4] Which calcium phosphate exchanges a fluoride ion for a hydroxyl ion, and what property is enhanced by fluoride? 190 MEMBRANES STRUCTURE 46. Which of the following compounds are bonded to phosphate in diacyl phosphoglycerol to complete the repertoire of phospholipids found in body membranes? A. Only choline B. Betaine and sphingosine C. Choline, betaine, and sphingosine D. Choline, serine, and sphingosine * E. Choline, serine, ethanolamine, inositol Related study questions [1] What are diacylphosphoglycerol, phosphatidyl choline, phosphotidyl serine, phosphatidyl ethanolamine, and phosphatidyl inositol?191 [2] Which amino acid is converted to ethanolamine, choline, and betaine; which sulfur-containing amino acid is salvaged in which betaine is involved?192 [3] What function(s) do phosphatidyl lipids serve in the membranes or the cytosol? 193 NERVOUS SYSTEM GENERAL PROPERTIES 47. The enzyme UNIQUE to nerve tissue is: * A. glutamate decarboxylase B. ornithine decarboxylase C. tryptophan decarboxylase D. glycine decarboxylase E. aspartate decarboxylase Related study questions [1] Which amino acid metabolism enzyme listed is tissue-specific for nerve tissue? 194 [2] What is the amino acid product of each listed enzyme and what vitamin/coenzyme is involved? 195 [3] GABA is an abbreviation for what amine derived from glutamate? 196


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[4] Where is GABA synthesized and stored and what is GABA used for?197 MUSCLE SKELETAL 48. Which comparison between hemoglobin and myoglobin of skeletal muscle is INCORRECT? A. Myoglobin is more oxygen saturated at lower PO2 levels than hemoglobin. B. Both are inside cells. C. Both contain Fe2+ combined with porphyrin. * D. Each molecule is composed of different subunit proteins. E. Hemoglobin transports oxygen while myoglobin stores oxygen. Related study questions [1] What are myoglobin and hemoglobin, what tissues contain them, what are their functions? 198 [2] What is the porphyrin family; where are they synthesized? 199 [3] What divalent metal cation does porphyrin bind in humans and what common name is given to the metalloporphyrin? 200 [4] What is the significance of the myoglobin O2 saturation curve being hyperbolic but hemoglobin’s is sigmoidal? 201 CIRCULATION COAGULATION 49. The coenzyme for γ−carboxylation of glutamyl residues required for synthesis of Ca2+ chelation clusters near the Nterminus of zymogens II, IX, X, and IX contains which vitamin? A. pantothenic acid B. B1 * C. K D. lipoic acid E. biotin Related study questions [1] What chemical properties does the γ-carboxyglutamyl side-chain have that enables it to act as a divalent metal cation chelation group?202 [2] What function results from the cluster of γ-glutamates near the N-terminus of specific coagulation zymogens? Why those four factors and not some of the others?203 [3] When trauma disrupts endothelial cells lining blood vessels thereby exposing phospholipids, factors, II, IX, X, and XI concentrate on the phospholipids exposed at the trauma site; what enzymological principles and effects on reaction rates are demonstrated?204 [4] What role(s) does γ-carboxyglutamyl residues in bone osteocalcin suggest for a role(s) of vitamin K in osteoporosis? 205 [5] Can you think of an example of carboxylations that are on the α−, β−, δ−, and ε−carbons of other metabolites? Which of these might act as chelators?206 RESPIRATION GAS EXCHANGE AND TRANSPORT 50. All of the following are true for oxygen carriage biochemistry EXCEPT one. Which is the EXCEPTION ? A. Binding of 2,3–bisphosphoglycerate to hemoglobin lowers O2 affinity for hemoglobin in capillaries. B. Association of H+ to hemoglobin decreases its affinity for oxygen. C. CO2 is a negative allosteric effector that promotes O2 dissociation from HbO2 when it forms carbaminohemoglobin. * D. Deoxyhemoglobin (reduced) has a lower pKa than oxyhemoglobin. Related study questions [1] What pathway in the red blood cell does 2,3-BPG arise from; how does the nearly molar equivalent ratio of 2,3-BPG: deoxyhemoglobin chains affect O2 binding?207 [2] Which has a greater affinity for H+: deoxyHb or HbO2?208 [3] Explain Bohr’s observation that increasing the CO2 in blood released oxygen from it. What other molecule is involved?209 [3] Where on the β-globin chain of Hb does CO2 react to form carbamino Hb; how much CO2 is transported as CO2, as HCO3–, as carbamino Hb in RBC, in plasma?210 EOF:gcl


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National Board Part I Examination Specifications (2002) Building Blocks of Life Course (41) I. Physical-Chemical Principles (3) A. Basic principles (2) B. Applied principles (1) II. Biological Compounds (9) A. Sugars and carbohydrates (1) B. Amino Acids and proteins (2 ) C. Lipids (1) D. Nucleic Acids and metabolism (1) E. Interdisciplinary clinical/cross correlation (4) III. Metabolism (13) A. Bioenergetics (1) B. Enzymology (1) C. Catabolism (2) D. Anabolism (2) E. Regulation (2) F. Interdisciplinary clinical/cross correlation (5)

---25 --IV. Molecular Biology (8) A. DNA/RNA and protein synthesis (4 ) B. Genetic engineering (2) C. Interdisciplinary clinical/cross correlation (2) XIV. Nutrition (8) A. Nutrients/minerals (3) 1. Requirements (1) 2. Functions (2) B. Interdisciplinary and clinical/cross correlation (2)

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---9 of 25 --Biochemistry in other D1 Courses (13) V. Connective Tissues (8) (in Basic Tissues) A. Soft tissue (2) B. Hard Tissue/calcification (3) C. Interdisciplinary and clinical/cross correlation (3) VI. Membranes (4) (most in Cell Organelles ) A. Structure (1) B. Function (1) C. Interdisciplinary clinical/cross correlation (2) VII. Nervous System (9) (most in Organ Systems) A. General properties (2) B. Central nervous system (1) C. Autonomic nervous system (1) D. Somatic nervous system (with reflexes) (2) E. Special senses (1) F. Interdisciplinary and clinical/cross correlation (2) VIII. Muscle (6) () A. Skeletal (2) metabolism and myoglobin B. Smooth (1) C. Cardiac (1) D. Interdisciplinary and clinical/cross correlation (3) IX. Circulation (9) (in Basic Tissues/clotting/gas transport A. Fluid content and dynamics (2)

2003 Biochemistry Review for the National Boards Part I ©2003 Gene C. Lavers, Ph.D. B. Coagulation (1) C. Cardiodynamics and electrophysiology (2) C. Regulation (1) D. Interdisciplinary and clinical/cross correlation (3) X. Respiration (6) (in Basic Tissues) A. Mechanical aspects (1) B. Gas exchange and transport (1) C. Regulation (1) D. Interdisciplinary and clinical/cross correlation (3) XI. Renal (6) (see Organ Systems) A. Functional anatomy (1) B. Blood flow and filtration (1) C. Reabsorption and secretion (1) metabolites D. Interdisciplinary and clinical/cross correlation (2) XII. Acid-Base Balance (1) (in Organ Systems)

Page 17 of 35 XIII. Digestion (5) (in Organ Systems) A. Neuromuscular (1) B. Secretions (1) enzymes C. Absorption (1) D. Regulation (1) E. Interdisciplinary and clinical/cross correlation (1) XV. Endocrines (8) (in Organ Systems) A. Pituitary/hypothalamus (1) B. Reproduction (1) C. Signaling systems (2) insulin/glucagon & cAMP D. Pancreas/parathyroid (1) E. Adrenal/thyroid (1) F. Interdisciplinary and clinical/cross correlation (2)

END NOTES – Answers to Related Study Questions

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The Biochemistry-Physiology Part I National Boards Examination covers 15 topic areas with 80 subtopics in 100 questions listed below divided into the Topics covered by Building Blocks of Life and other D1 basic science courses.

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Plasma pH 7.45, although stomach (3.5), duodenum (8.0), etc may differ. The strength of an acid is determined by (a) the strength of its conjugate base, (b) the basic strength of the solvent, and (c) the dielectric constant of the oppositely charged particles dissolved in them, which necessarily favors dissociation. In aqueous solution, strong acids completely ionize (e.g., HCl, because HOH, water is a weak acid), whereas weak acids (acetic acid in water) ionize to the extent of the density of negative charge on the conjugate base (CH3COO–). Iodoacetic acid is stronger than acetic acid because iodine on C2 inductively weakens the acid bond and decreases the carboxylate anion negative charge density, hence, protons dissociate more easily and are less attracted to the weaker iodocarboxylate anion (ICH3COO–). compared to the acetate ion. This phenomenon is called the neighboring group effect. Substitution of an amino group on C2 (instead of Iodine) yields glycine, the –NH3+ thereby inductively makes the –COOH a stronger acid than the –COOH of acetic acid so the pKa of former is lower than acetic acid’s. Of those listed, only D. –COOH, carboxylic acid group is dissociated at pH 7.5. A. CH2 methylene hydrogens, B. C–OH alcohol group, C. –NH3+ ammonium, E. phenol all are not dissociated at pH 7.5 to any significant degree, which is what the question asked. However, strong electron withdrawing, neighboring groups (e.g., in malonyl CoA) may lower the pKa sufficiently close to pH 7.5, for the –CH2– middle methylene to be significantly ionized –OOC–CH2–CO-CoA yielding a carbanion –OOC–CH(–)–CO-CoA. In the ionized zwitterion (–/+)of an amino acid, the protonated –NH3+ ammonium group strongly attracts electron density from the ionized –COO– anion making it a weaker conjugate base (less electron dense), hence the proton dissociates more readily then an isolated carboxylate anion (e.g., as in acetate). Conversely, the electron density available on the –COO – can be donated to the electron deficient (positively charged) amino group thereby lessening its electron deficiency. The mutual neighboring group effects shift the pKa of each group by about 1-2 pH unit.

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Two, analogous to a pair of left and right hands. Using Fischer representation, when several asymmetric carbon (C*) atoms are in a chain molecule, and the end groups are not identical and not asymmetric, the remaining number of stereoisomers possible is equal to 2(exp n), or 2n, where n is the number of asymmetric carbon atoms. When linear sugars cyclize, an additional C* is formed (because the C 1 aldehyde reacts with the internal alcohol (to form an intramolecular cyclic or internal hemiacetal, which is a new asymmetric center). [Note: a hemiacetal can condense with a second mole of alcohol to form an acetal, e.g., D-glucose + methanol  methyl–α− and –β−D–glucoside, same number of isomers are possible.] For linear aldopentoses: 2(3) = 8; for aldohexoses: 2(4) = 16. For ring-closed (Haworth projection) pentoses: 2(4) = 16, for hexoses: 2(5) = 32. For linear ketohexoses: 2(3) = 8. [C2 ketone the internal keto group and two non identical end groups leave only 3 C*. For closed-ring hexose: 2(4) = 16. [C2 ketone adds an alcohol to form an intramolecular cyclic or internal hemiketal, which is asymmetric (can form 6- and 5-membered rings). [Note: a hemiketal condenses with an alcohol to form a ketal, e.g., Dfructose + methanol –> methyl–α− and –β−D–fructoside, same number of isomers possible.] C1 is the anomeric carbon of aldose sugars. Hemiacetal formation creates a new C* center, thus two anomers are formed: α− and β−D-aldose sugars, e.g., linear D-glucose cyclizes –>cyclic α−D-glucose (+112°) and cyclic β−D-glucose (+19°), an anomeric pair with a 1:2 ratio of α:β forms (+52.5°) [Note, racemic pairs have a 1:1 ratio, they have no net optical rotation.] An epimer is a pair of sugars that differ at only one asymmetric carbon, e.g., D-glucose and D-mannose have opposite configurations at C2 and glucose and galactose differ only at C4. In Fischer projection, the bottom most C* center of all sugars relates to C2 of glyceraldehyde and defines the sugar as D or L. [The sole C2* of glyceraldehyde (simplest aldose sugar, also named glyceric aldehyde) exits in two stereo isomer forms: C2–OH (on right side) defines D-form, (left) HO–C2 defines the L-form. All the D-aldose sugars (4-, 5-, 6-, 7carbons) are considered derivatives of D-glyceraldehyde by adding one CH2OH at a time; all L-sugars are mirror image molecules and are derived from L-glyceraldehyde.] Each C* center is asymmetric and rotates polarized light left or right depending upon which one of the configurational isomers is being measured, if both are present, racemate pair (1:1 ratio), no net rotation occurs (unless another structural influence shifts the ratio higher or lower). Reference glyceraldehyde: D-glyceraldehyde rotates polarized light rightward (dextro, [α]D = +13.5°) and Lglyceraldehyde rotates polarized light leftward (levo, [α]D = –13.5°). More complex sugars, aldopentoses and aldohexoses may give different rotations depending upon the summation of all the C* ( – or + ) rotations. [ Note: dextro and levo are also notated as, d and l, respectively, e.g., l-lactic acid or d-lactic acid. No, the “D” and “L” prefixes have no relationship to the direction of optical rotation. D and L simply designate spatial relationship of the groups on the penultimate C* of the compound in reference to the D and L forms of glyceraldehyde.

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In a meso stereoisomer, two halves of the molecule are mirror images. Such a diastereomer is uniquely optically inactive; the mirror halves constitute a racemic mixture, thus the optical rotation of one halve is reversed (cancelled) by the second half. The simplest example of a meso versus diastereoisomers comparison is tartaric acid (2,3-dihydroxy-1,4-succinic acid), which has two middle C*s. In Fischer projection, the top and bottom carbon are identical (COOH). The configuration of the –OH group on C2 and C3 are: left-right (I), right-left (II), and left-left (same as right-right), respectively. Form I is levo- or l- or D-tartaric; form II is dextro- d- or L-tartaric, and form III is meso tartaric or m-tartaric. Living systems operate at constant temperature (isothermally) and constant pressure (isobarically) and cannot convert heat energy directly into work as an engine does. Enthalpy (H) is the heat energy consumed or released in a system at constant pressure. Entropy (S) is the energy that is unavailable to do work and is lost to disorderliness of a system, entropy increases as temperature increases. Free energy (G) is the energy of a system that is available to do work, at constant temperature and pressure, so it is useful in studying the bioenergetics of living systems. Activation energy (Eact) is that amount of energy molecules most possess before they are capable of reacting. Potential Energy is the energy of a molecule due to its position. Only the change (∆) in H, S, Eact is measurable and has practical significance. [Potential energy is converted to Kinetic energy while the molecule is moving from its original position to another. As a ball rises in the earth’s gravitational field, kinetic energy is converted to potential energy – maximal at the instant the ball is suspended –before descending; while descending, the potential energy is converted to kinetic energy (motion]. The more random disorder (the higher the entropy, S) in a system, the less energy is available to do work. Live systems expend energy to organize molecules into highly organized functional structures and systems (membranes, filaments, organelles, DNA sequences of specific order, etc.). Reversible reactions rarely reach equilibrium in vivo so entropy is kept lower than at death, whereupon all equilibrium reactions tend towards equilibrium, and entropy is maximized. The energy used to create orderly DNA sequences is released as DNA is hydrolyzed to disordered nucleotides moving with Brownian motion in solution. The probability of the nucleosides randomly reorganizing back into the original ordered sequence is highly improbable, the energy lost to entropy prevents it. Spontaneous regeneration of life from thermodynamic death is virtually improbable, although, theoretically it is not entirely impossible. Assembly of monomer units into polymeric sequences increases orderliness and decreases entropy, hence glycogenesis, FA synthesis, and the synthesis of proteins, DNA and RNA have lower entropy than their corresponding monomers. Assembly of monomers decreases entropy, disassembly of polymers increases entropy. Therefore, degradation of glycogen, fatty acids, proteins, DNA and RNA to glucose, acetyl CoA, amino acids, deoxyribonucleotides and ribonucleotides, respectively, decreases orderliness and increases disorderliness, so entropy increases during the processes of glycogenolysis, β-oxidation, proteolysis, and hydrolysis, respectively. Nucleophilic attack by an alcoholic O: (or, amino N: , mercapto S: ) from above or below a planar, trigonal, electron deficient C of an aldehyde (R–CH=O, or ketone RR'–C=O) forms a tetrahedral product. Four different chemical groups are bonded to the anomeric C in the hemiacetal so it an asymmetric (C*). If attack from below or above is equally probable (no structural restraints) then a pure 1:1 ratio of enantiomeric isomer products forms. In aldoses such as glucose, the C5– ÖH attacks intramolecularly yielding a stable 6-membered ring (Haworth diagram) held by a hemiacetal linkage. C1 is bonded to four different chemical groups, is asymmetric (C*). Ignoring the reaction mechanism, the aldehydic C=O becomes C1–OH in the hemiacetal group. Attack from above yields the α-anomeric configuration (α-OH) under the ring, in Haworth projection); attack from above yields the β-OH anomeric configuration. Refer to study answer to Question 2[1]. In forming the intramolecular ring, the aldoses are expected to gain an additional asymmetric carbon (optically active) in the resulting hemiacetals and hemiketals. Thus, aldose D-glucose (4C*) ring closes –> α−D−glucose (5C*) + β−D−glucose (5C*); and ketose D-fructose (3C*) –> α−D−fructose (4C*) + β−D−fructose (4C*). D-ribose (3C*) –> α/β-D-ribose. The process of mutarotation opens and closes the ring of sugars again and again. If freshly dissolved pure α (or β), is observed through a polarimeter, initial degrees of polarized light rotation slowly changes until an unchanging reading is achieved, i.e., an equilibrium mixture of α/β anomers is present. [However, in α-glucose, the OH groups are crowded together (axial, flagpole interactions) above and below the ring; in the β-anomer, these bulky groups point outward in the plane of the ring (equatorial) so steric crowding is minimal, the β-glucose is more stable (lower free energy). For glucose, a pure 1:1, α/β, racemate mixture is not formed, instead a 1:2 (α/β) ratio is established at equilibrium.] Closed-ring hemiacetals and hemiketals react with hydroxyl ions in solution, at physiological pH, thereby reverting back to the aldehydic (–CHO) or ketone (C=O) ring-open form; thus, OH– ions catalyze the mutarotation rate upwards of about 40,000-fold! In contrast, acetals and ketals are very stable to OH– ions (because the anomeric carbon is linked to an –OR group not an –OH). Hence, disaccharides and polymers with α-acetal/ketal linkages are very stable (don’t hydrolyze readily and are excellent for storage of glucose fuel (glycogen, starch). Similarly, β−acetal linkages (cellulose in wood)

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are stable and strong polymeric molecules. [Note: in a polymer (starch, glycogen, or cellulose), the end glucose residue has a free hemiacetal or ketal group and can mutarotate, while all the other glucose residues in the polymer will not.] Common table sugar, sucrose is an acetal/ketal combination: glucose-α1–β2-fructose; both anomeric carbons are in the one joining linkage, this disaccharide doesn’t mutarotate! Yes, the nucleoside components contain N−β−D–ribose in an N-acetal linkage to adenine, cytosine, guanine, or uracil in RNA, and N−β−D–deoxyribosides of adenine, cytosine, guanine, or thymine in DNA. Direct structural correspondence of L-amino acids to L-glyceraldehyde. Some bacteria and antibiotics use D-amino acids. See Fig 2.3 in Baynes and Dominiczak, Medical Biochemistry, for a list of structures, then Fig. 2.5 for those that are acidic or basic according to the side-chain functional group. Which amino acids have acidic carboxyl –COOH; basic amine –NH2; imidazole (histidine :N-ring), guanidino (arg). Also see Fig 2.7 for conjugate acid/base forms and pKa values.) Amino acids are amphoteric molecules, they have at least one basic and acidic group. Upon dissolving glycine in water, for example, the α-NH2 amino group (pKa 9.8) becomes protonated to –NH3+ ammonium; the α−COOH (pKa 2.4) ionizes to –COO– ; both ionic groups define a zwitterion (double ion) with net charge (0) at pH7.5. [ ] For the Asp and Glu, the side-chain –COOH (pKa 3.9) ionizes yielding a net (–1) charge at pH 7.5. For lys, his, an arg, these become protonated yielding a net (+1) charge at pH 7.5. At pH 2 all amino acid carboxyl groups are COOH, all basic groups are protonated. Depending on the pKa of each group, as hydroxyl ions are added, the pH changes from 2 to 12, each acid and base group is titrated in order of their increasing pKa value. At pH 12, all acids groups are COO– and all protonated bases are now in free-base form (e.g., – NH2). At pH 2, most amino acids move to the anode, each will not move when the pH equals their isoelectric point, as pH becomes increasingly basic, the amino acids will begin to move to the cathode, respectively. See Fig. 2.6 in Baynes for the titration curve describing the progression of cation to zwitterion to anion of alanine, as an example of a simple amino acid. Some amino acids have a third acid or basic group on their side chain. Their titration curves are slightly more complex, but manageable). [Electrophoretic migration of the 20 amino acids is pH-dependent on either side of their pI isoelectric point: (a) simple amino acids: asn 5.41, gln 5.65, ser 5.68, met 5.75, gly 5.97, val 5.97, leu 5.98, phe 5.98, ala 6.02, ile 6.02 (2 C*), thr 6.53; (b) acidic amino acids: asp 2.97, glu 3.22; (c) basic amino acids: lys, 9.74, arg 10.76; (d) other ionizable side chain groups: cys 5.08, tyr 5.65, trp 5.88, pro 6.10, his 7.58. Again, lower acidity moves them to the (−) anode, higher to the (+) cathode. The pI is the average of the 2 or 3 pKa values. The (a) group give dipolar zwitterions between pH 3 and 8 and barely migrate except at their isoelectric point (zero net charge, no movement). At physiological pH, the net charge of free amino acids and when in a polypeptide is given for reference: At pH 7.5, free amino acids, net charge (0): asn, gln, ser, met, gly, val, leu, phe, ala, ile, thr, his, cys, tyr, trp, pro two (−1): asp, glu two (+1): lys and arg. At pH 7.5, within a polypeptide, zero charge (0): asn, gln, ser, met, gly, val, leu, phe, ala, ile, thr cys, tyr, trp, pro (−1): asp, glu, his (+1): lys, arg. ]

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The 20 amino acids given in footnote 28 are always incorporated into proteins during synthesis of their primary structure. Specific amino acids (13) undergo posttranslational modification in certain proteins (see Fig 2.3) so the number and variety of such modifications is complex. Methylations (lys, arg), phosphorylations (ser, thr, tyr), carboxylations (glu), disulfide oxidations (cys) amidations (asp, asn, glu, gln), and hydroxylations (pro phe, tyr) are some commonly encountered post translational modifications. Some pathways create/consume other amino acids, e.g., ornithine, citrulline, argininosuccinate (urea cycle), homocysteine (from S-adenosyl methionine), β-alanine (pyrimidine catabolism), GABA (from glu), and many others.

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A very complex subject. See Fig 2.12 in Baynes for four major sets of side-chain interactions. The 3D-folding of proteins with more than about 200 amino acids consists of several smaller folded units designated as domains such as alpha-helix, pleated-sheets, random coil, and others. These and other 3D tertiary (3°) structures of a protein are stabilized by sidechain functional groups: covalent sulfhydryl bonds (cys-S—S-cys), hydrogen bonds (O–H•••N), salt bridges (–COO– •••H+NH2–) and hydrophobic interactions (e.g., phe-phe, leucine zippers, zinc fingers, etc.). (See Baynes Ch 2 under Tertiary Structure, Quaternary structure headings). Solvent effects, influenced by salts, and other substances can be important. Some or all of these properties can be involved in quaternary structure, e.g., tetrameric hemoglobin (α2β2) The nonpolar hydrophobic amino acids (gly, ala, leu, ile, val. phe, met, etc.) associate in hydrophobic pockets inside the protein, water is excluded into the surrounding solution. The polar hydrophilic and acid base side-chain amino acids (are usually on the surface in contact with water, also anions and cations of a wide variety. Some domains are folded to expose

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a hydrophobic surface (e.g., membrane proteins, leucine zippers). Glyco- and lipoproteins can have sugars and lipids bonded to the side-chains of ser, thr, try, hyp, lys, etc.). In Baynes, see Fig 17.2; 24.1, 2, 3, 4; 26.4, 6, 12. Classification: (a) Fatty acids are long chain hydrocarbons with a terminal carboxylate (not found free but always in ester linkage with triple alcohol glycerol because free fatty acids are soaps and would dissolve cell membranes and organelles) or are a in thioester linkage with CoA, ‘activated’ fatty acid, (b) triglycerols (fuel energy) are most abundant, [also waxes: fatty acid esters of alcohols other than glycerol], (c) phospholipids (membrane structures; derivatives of glycerol phosphate, diesters most abundant, second, derivatives of sphingosine phosphate, which contain an esterified fatty acid (FA) and a nitrogenous base (choline, ethanolamine) which may also be esterified with a phosphate. (d) Nonphosphorylated lipids (1. cerebrosides, glycolipids: derivatives of sphingosine having a FA and hexose substituent. 2. Sulfolipids: derivatives of sphingosine, FA and sulfated hexose substituent. 3. Gangliosides: derivatives of sphingosine, FA, hexosamine, hexose and sialic acid. 4. Proteolipids: complexes of lipid and protein. 5. Steroids: derivatives of cyclopentanoperhydrophenanthrene. Triacylglycerol or triacylglycerides or triglycerides are idea as Fuel: because the fatty acids are essentially polymerized hydrocarbon, (CH2) molecules, that metabolic oxidation converts to CO2 and about 9 kcal/gram of FA, the highest caloric value compared with carbohydrates and proteins (both about 4 kcal/gram). Solubility: extractable by ether, chloroform or alcohol. FA less than C6 are soluble in water (e.g., acetic – butyric acids). Density: as low as 0.7 g/ml makes them lighter than water. Melting points: 1C to 9C are below body temperature, 10Cs and more are likely solids; cis C=C bonds lower mp enough to be liquid oils at body temperature – that’s important for membrane fluidity. Insulator: effective thermal and electrical. Padding/supporting: the kidneys are surrounded by lipid fat. Phospholipids form bilayer sheets in water, and if shaken will form spherical bilayers filled with solvent (water) called micelles, which are a primitive model of a cell or cell organelles that serve to separate (partition) molecules and structures with different compositions and functions. Further considerations of lipids and membranes are taken up in Cell Organelles Course by Prof. Roy, refer to your notes. Phospholipids (or phosphatides) are a heterogeneous group of compounds found in virtually every living cell and may be the chief cellular lipid component. In general, natural phospholipids have the L-configuration, so phosphatidic acids contain L–α– phosphatidic acid with two FA esterified to the β and γ-carbon of glycerol. The phosphate may be esterified to choline (lecithins), ethanolamines (cephalins), both are the most abundant (phosphatidyl-inositol, -serine, -glycerol, also occur). In contrast, sphingolipids have a unique core molecule, sphingosine, D-erythro-1,3-dihydroxy-2-amino-4trans-octadecen (18 carbon chain length). Hydrolysis of sphingomyelins yields sphingosine, phosphate, a fatty acid, and a nitrogenous base (mostly choline, although ethanolamine is found too). The relationship between the H, A, and B blood-group substances (see Fig 25.15 in Baynes) relates to the terminal oligosaccharide linked via other sugars to proteins and lipids on the red cell membrane. The H-substance has protein and sphingolipid attached. Individuals with type A blood designation have Gal-N-Ac-α1,3 attached to the galactose of Hsubstance to form the A-type glycolipid. Type B have Gal-α1,3 to the galactose of H-substance to form the B-type glycolipid. Type AB have both Gal and Gal-N-Ac attached to the galactose of H-substance. Type O have neither sugars added to the galactose of H-substance. In each blood type: A, AB, and B, a specific transferase gene is expressed that adds the specific sugar(s); neither gene is expressed in type O individuals, who lack both enzymes. See Fig 28.3 in Baynes. Atoms incorporated onto purine ring of IMP derive from : N9-glutamine, C4,5 N7-glycine, C8-N10formyl THFA, N3-glutamine, C6-CO2, aspartate, C2-N10-formyl THFA yields hypoxanthine ring of inosine-5P (IMP). See Fig 28.4 in Baynes. AMP <– adenylosuccinate <– IMP –> XMP –> GMP. Asp is used to make AMP; NAD+, then gln are used to make GMP. Specifically: Rxs 11. IMP + asp + GTP5 –> adenylosuccinate (similar to argininosuccinate in urea cycle) 12. adenylosuccinate –> AMP + fumarate 11. IMP + NAD+ –> XMP + NADH 12. XMP + gln + ATP5 –> amide (rearranges) to imide of guanine in GMP. Five specific steps involve coupling to ATP hydrolysis for synthesis of an amide in each case. Amides require high energy input usually obtained from coupled to ATP hydrolysis. The following 12 steps review purine de novo synthesis. The four for hypoxanthine and ATP or GTP coupled reactions for AMP and GMP are bold. Five total for AMP and GMP. 1. PRPP + Gln –> 5P-ribosyl-1-amine + glu + PPi 2. 5P-ribosyl-1-amine + gly + ATP1 –> glycinamide rt + ADP + Pi [note: 5P-ribosyl = ribonucleotide = rt] 3. glycinamide rt + N10-f-FH4 –> formyl-glycinamide rt + FH4 4. formyl-glycinamide rt + gln + ATP2 –> f-gly-amidine rt + glu + ADP + Pi [amidine is a 2N amide, C=N substitutes for C=O]

5. f-gly-amidine rt + ATP3 (ring closure) –> 5-NH2-imidazole rt +ADP + Pi [closed-ring amide that rearranges to imidazole] 6. PR-5-NH2-Im rt + CO2 –> 5-NH2-4-carboxyate-Im rt 7. 5-NH2-4-carboxylate-Im rt + asp + ATP4 –> 5-NH2-Im-4-N-succinocarboxamide nt + ADP + Pi 8. 5-NH2-Im-4-N-succinocarboxamide nt –> 5-NH2-Im-4-carboxamide nt + fumarate 9. 5-NH2-Im-4-carboxamide nt + N10-f-FH4 –> 5-f-NH2-Im-4-carboxamide rt + FH4 10. 5-f-NH2-Im-4-carboxamide rt + H2O –> hypoxanthine rt = inosinate = IMP 11. IMP + asp + GTP5 –> adenylosuccinate (similar to argininosuccinate in urea cycle) 12. adenylosuccinate –> AMP + fumarate 11. IMP + NAD+ –> XMP + NADH 12. XMP + gln + ATP5 –> amide (rearranges) to imide in guanine of GMP. 41

N10-formyl-THFA supplies C8 and C1 in hypoxanthine in IMP synthesis; CO2 supplies C6 of IMP; SAM for GpppNmRNA –> m7GpppN-mRNA, other RNAs and proteins.

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Glutathione (GSH) is L-γ-glu-cys-gly [note the side chain –COOH of glu is bonded to cys]. The cys-SH is the active antioxidant functional group, it supplies electrons thereby preventing other molecules (proteins) from being oxidized (loss of electrons brought about by interaction with reactive oxygen species. By donating electrons GSH becomes oxidized instead and it forms dimer GSSG. There are two tissues that must deal with high levels of oxidative insult that are highly differentiated, the red cell and the eye lens, both have GSH levels between 6-8 mM. Red cells are constantly exposed to high levels of oxygen. In contrast the pO2 in lens is so low (oxygen diffusion from vascularized retina) that it can’t be accurately measured. Lens oxidation is due to photooxidation by ultraviolet radiation. Since visual clarity must be maintained, and all lens proteins are present throughout life (no turnover), antioxidant protection by GSH is absolutely essential to prevent cataracts (opacity, due to light scattering by oxidized proteins and other physicochemical effects). In Baynes, last topic, “A small fraction of triose phosphates produced during metabolism spontaneously degrades to methylglyoxal” CH3-CO-CHO, adjacent, highly reactive aldehyde-ketone groups “methylglyoxal (MG) reacts with amino, guanidino (arg), imidazole (his) and sulfhydryl (cys) groups in proteins, leading to enzyme inactivation and protein crosslinking. Methylglyoxal is also formed during metabolism of acetone (CH 3-CO-CH3) and glycine. The glyoxalase pathway (Fig 11.15) is a 3-Rxn pathway to detoxify methylglyoxal to D-lactate. MG and GSH form a hemithioacetal (pyruvate-SG) that glyoxalase I rearranges to D-lactate-SG that glyoxalase II hydrolyzes to D-lactate and releases GSH to be used again, i.e., MG + GSH –> MG-SG (glyoxalase I) –> Pyr-SG (+ H2O, glyoxalase II)–> D-lactate + GSH Oxygen is the indispensable final recipient of metabolic electrons. When reduced by cytochrome a3 to water all latent energy in the larger perspective that is derived ultimately from photosynthesis has been converted to free energy to drive the body, any energy difference is lost to other thermodynamic energies, such as heat and randomness, relate to the efficiency of the bioenergetics of life processes. Any diminution of oxygen supply entering the mitochondria proportionately diminishes the entire oxidative metabolic apparatus, which rapidly manifests as hypoxia in all vascularized tissue cells, especially the brain. Molecular oxygen is relatively inert, but is a strong oxidizing agent. Iron heme and membrane lipids are easily oxidized. All oxidases and oxygenases (metalloenzymes) use molecular O2 and produce partially reduced reactive O2 species (ROS) such as oxygen superoxide radical anion (O2•– ), its protonated form (hydroperoxy radical HOO• , pKa ≈ 4.5), and hydrogen peroxide (H2O2) that are more reactive then O2 and are precursors to strongly oxidizing species such as hydroxyl radical (OH• ) and metal-oxo complexes (Baynes Fig 11.10). Functional hemoglobin (Hb) contains ferrous iron (Fe2+) which binds O2. Hb can spontaneously produce O2•– in a side reaction associated with O2 binding that converts hemoglobin (Fe2+, red) to methemoglobin (Fe3+, reddish brown). MetHb may precipitate in the RBC, forming inclusions known as Heinz bodies, and may also release heme, which reacts with O2•– and H2O2 to produce OH• and reactive iron-oxo species. [ROS species form lipid peroxides that decompose to reactive carbonyl species that react with proteins, damaging the integrity of the cell membrane and the activity of transporter proteins, collapsing ion gradients and leading to cell death.]

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Both separate salvage Rxns uses PRPP to supply ribose-5P: Enz: adenine-phosphoribosyltransferase: ade –> AMP; Enz: hyp-gua PRtase: hyp or gua –> IMP or GMP. Both enzymes convert free base to nucleotide in one step. The most prevalent deficiency (HGPRTase) results in excessive oxidative loss of hyp and gua to uric acid that presents as hyperuricemia. The consequences come from the sparing solubility and needle-sharp sodium uricate crystals. These most easily appear in the cooler joints of the hands and feet leading to acute gout-like episodes beginning in middle age in men. If brain levels of HGPRTase below 1%, uncontrollable psychiatric, self-mutilation occurs, with life-span limited at best to teenage years if untreated chiefly due to renal failure.

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Xanthine oxidase (XO, a molybdenum (Mo6+-iron-containing flavoprotein) oxidizes hypoxanthine –> xanthine –> uric acid using molecular oxygen (which is reduced to H2O2 , then decomposed to water and O2 by catalase). Once XO acts on allopurinol as a substrate, its oxidation product, alloxanthine (oxipurinol), binds strongly to XO, inhibits the enzyme, hence the designation as a suicide inhibitor. Allopurinol is an analog of hypoxanthine in which C8 and N9 are switched the two ring N’s next to each other. [Allopurinol prevents Mo4+ reoxidization back to Mo6+ (active enzyme). With XO inhibited by allopurinol, PPRP levels are higher in gout (may approach normal levels) salvage operates more normally so free ade, hyp, and gua bases go back into AMP, IMP, and GMP nucleotides for continued usage rather than oxidative loss to urate. Without allopurinol, purine base levels decrease and insoluble uric acid increases (hyperuricemia). Additionally, the high PRPP levels stimulate more de novo purine nucleotide synthesis that leads to further uric acid increases (in Lesch-Nyhan syndrome of up to 50 mg urate/kg body weight/day) above the normal output of 10 mg urate/kg body weight/day. ATP hydrolysis. Creatine phosphate (creatine-P). This secondary, high-energy reserve (∆Gº'CP = – 12.0 kcal/mol compare to (∆Gº'ATP = – 7.3) converts ADP to ATP + creatinine. The phosphate of creatine-P is in a very high-energy bond (∆Gº'CP = – 12.0 kcal/mol). The quanidino group of creatine (from arg) makes an N-anhydride (similar to O-acid anhydrides, e.g., acetyl anhydride, carbamoyl-P), all of which have a high (∆Gº') of hydrolysis. When the creatine-P cyclizes, the attacking COO (from the other end of the molecule) forms an cyclic amide and the stable leaving group, Pi, leave sufficient free energy to phosphorylate ADP. Creatinine is the cyclic N-anhydride product of creatine-P, it has no other metabolic use and is excreted from the plasma into the urine. Anaplerosis reactions convert metabolites to TCA cycle intermediates to replace those diverted out into other synthetic pathways (e.g., succinyl CoA into heme synthesis). The most immediate reaction supplies OAA (from pyruvate via pyruvate carboxylate), which together with pyruvate starts the cycle and ends by regenerating OAA. The next anaplerotic enzyme (glu transaminase) converts glutamate –> α-ketoglutarate; then malic enzyme converts cytoplasmic pyruvate –> malate which enters the mitochondrion. Thus, anaplerosis ensures that OAA is available for continued TCA function and also ensures that gluconeogenesis can occur using the carbon skeletons of glucogenic amino acids (when glucose supplies are diminished). Without PCase to convert pyruvate to OAA, pyruvate is converted to lactate, both accumulate. Increased cellular lactic acid leaves and enters the plasma acidifying it, i.e., causing lactic acidosis (one type of metabolic acidosis). Without PCase, high pyruvate leads to increased acetyl CoA via pyruvate dehydrogenase, which then funnels into fatty acid synthesis. Gluconeogenesis is inhibited, so the increases pyruvate goes to acetyl CoA increasing lipogenesis significantly. The pentose shunt supplies the NADPH. [Note, if gluconeogenesis is inhibited, then glucose is available to enter the pentose shunt yielding 2 NADPH per glucose and the carbons reenter glycolysis to give even more pyruvate, hence, acetyl CoA for continued lipogenesis.] The free energy content of a substrate (or its product) cannot be measured directly, only the difference or change (∆G) of free energy content between substrate and product can be measured. Enthalpy change (∆H) in heat gained or lost among the molecules of a reaction system, at constant pressure, decreases or increases the temperature of the reaction system surroundings (water, cytosol, mitosol, nucleosol, etc). The biochemical apparatus of the cell cannot convert heat per se into work, as a mechanical engine can. Thermal homeostasis mechanisms dispose of the heat through respiratory, circulatory, and perspiration mechanisms but most useful metabolic work would not occur otherwise (a hibernating bear). American chemist, Willard Gibbs developed the thermodynamics of free energy which relates the heat gained or lost in a reaction at constant pressure (isobaric) but reconciled it with the condition of constant temperature (T = is constant, isothermal), which are the conditions of biochemical reactions in animals with thermal homeostasis regulation. Hence, ∆G = ∆H - T∆S took into account the unusable difference in (∆H) heat as related to changes in system randomness (∆S) at a particular temperature. What was left was ∆G, energy free for useful physico-chemical work or achievement. Add algebraically Rx II + Rx I = (– 7.3) + (+ 3.3) = – 3.0 kcal/mol, the net energy difference is exergonic for glucose phosphorylation by hexokinase (or glucokinase). Exergonic: Rx I; endergonic: Rx II. Catabolism is exergonic; anabolism is endergonic; overall metabolism (catabolism + anabolism) is exergonic. A double reciprocal plot graphs 1/x versus 1/y. The Lineweaver-Burk plot graphs 1/[S] versus 1/V, respectively, and is advantageous, for the usual hyperbolic curve of the typical Michaelis-Menten plot is thereby linearized. The y-intercept gives 1/Vmax, the x-axis gives –1/Km. Simple calculation gives Km and Vmax.

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The Km is the substrate concentration ([S]) at which the reaction rate is half the maximal velocity (V max/2). As [S] increases, reaction rate (V) increases and approaches the maximal velocity possible, Vmax, asymptotically (for the amount of enzyme present, if the amount of enzyme doubles the rate doubles). Km is also a measure of the affinity of the substrate for the enzyme. The initial velocity (v o) gives the most accurate experimental measure of rate at the initial [S], which is known most accurately. Intermediate v values are more difficult to determine because of experimental uncertainties. In reference to the Michaelis-Menton hyperbolic curve, the section of the rate curve where [S} is below the Km is first order because the rate is proportional to [S]. The part of the rate curve corresponding to more than 5- or 10-times [S] above the Km is demonstrating zero order kinetics because the enzyme is working as fast as possible, independent of [S], because S is so abundant that any further addition of S does not increase the reaction rate significantly. Glycolysis is the central metabolic pathway for oxidizing glucose to smaller carbon fragments that have diverse anabolic and catabolic fates.

Anaerobic glycolysis also yields 2 net ATP, while aerobic glycolysis also yields 8 net ATP. are produced, 2 ATP are consumed . It serves as a preparatory pathway for either completing the oxidation (TCA) or storing the carbons in fatty acids (FA synthesis) as a depot fuel, or other uses (cholesterol, etc). When operating anaerobically it recovers only about 5% of the energy available from glucose that can be extracted, when operating aerobically, reduced NADH, produced by glycolysis, is oxidized with oxygen involvement (aerobically) in a mitochondria to extract nearly 40% of the energy in glucose. 64 Phosphorylation of F6P to F16BP by PFK-1 commits irreversibly the 6-carbon skeleton to oxidative glycolysis. The phospho-sugars in reactions preceding fructose-6P can flow into other pathways, so their carbon skeleton is not committed to glycolysis. Once F6P is acted upon by PFK-1, the only fate of F16BP is cleavage by aldolase and further glycolytic oxidations of each 3-carbon fragment to pyruvate (or lactate). 65 Pyruvate is the major product of aerobic glycolysis, lactate is the major product of anaerobic glycolysis. ATP is also a major product of both glycolytic process, anaerobic glycolysis yields a net of 2 ATP whereas aerobic glycolysis yields a net of 8 ATP (due to mitochondrial participation). [Both glycolytically produced NADHs have their electrons shuttled into the mitochondrion to yield an additional 6 net ATP]. and the pyruvate carbon skeleton is further oxidized in the mitochondrion to 3 CO2 and an additional 15 ATP are produced from reduced coenzymes 66

Lactate dehydrogenase (LD) prevents metabolic collapse by recycling the catalytic amount of NADH to NAD + so glycolysis continues regenerating ATP from ADP. This is made possible by reducing pyruvate (ketone) to lactic (alcohol). 67 Mitochondria. 68 Pyruvate kinase phosphorylates ADP to ATP using the high –∆G of PEP hydrolysis of the enol that yields pyruvate. 69 Anaerobic glycolysis (yields net of 2 ATP per glucose) versus aerobic glycolysis (yields net of 8 ATP per glucose). 70 The red blood cell can only produce glycolytic lactate! It lacks mitochondria! Hence, the RBC is a continuous, major source of plasma lactate. As the simplest of cells, the net 2 ATP yield is sufficient to meet RBC metabolic energy needs because cell’s role is transporting and delivering oxygen is maximized by having no need for oxygen. 71 The muscle cells constitute the major tissue mass of the body. When working anaerobically produce massive amounts of lactate which enters the plasma during strenuous exercise. 72

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Free glucose and glucose released from polymeric carbohydrates (starches) in foods are absorbed by the intestines and transported via the portal vein into the liver. Glucose utilization requires phosphorylation. Hexokinase has low Km for glucose and is G6P product inhibited, which is adequate for muscle cell glycogenesis and glycolysis needs, but inadequate for liver glucose metabolism needs, which must supply its own needs as well as supply the brain and all other tissues. Unlike muscle cells, liver cells also have hexokinase isozyme IV (glucokinase), which has better kinetics: high Km and no G6P product inhibition so G6P flows easily and rapidly into liver glycogenesis and glycolysis. At rest after a meal, a 70 kg man will store 200 g of glucose as liver glycogen and 150 g glucose as muscle glycogen, but overnight only 80 g of glucose in liver glycogen will remain. The other 120 g of glycogen glucose was released by the liver to supply the brain and other tissues with glucose overnight. During waking hour activities, additional amounts of glucose is oxidized by the liver for varied metabolic needs. Gene expression of the liver glucokinase gene is induced by higher than usual dietary glucose loads if sustained over several days thereby enabling the liver to adapt to extraordinarily elevated dietary glucose and higher than usual hyperglycemia. The other enzymes listed in the question are part of glycolysis.

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The acetal functional group (C bearing two OR groups) defines O-glycosides (e.g., glycogen and cellulose). It contains two stable ether-like bonds (C–O–Ca and C–O–Cb) on the anomeric C* so glycosides are stable, it will not hydrolyzed on its own. One OR is in the sugar ring, the second OR is a linked sugar residue (replaces H in the hemiacetal of free glucose). There are two possible stereochemical isomers (α and β at the C*) for the O-sugar: either above or below the ring, respectively. Glycoside synthesis and hydrolysis enzymes are stereospecific for α- or β-glycoside anomer linkage, i.e., α1,4 (starch, amylase) and α1,6 (glycogen, debranching enzyme, then amylase) or β1,4 (cellulose, cellulase). Dietary starch (fuel) digestion begins in the mouth with salivary α−amylase, later in the gut with pancreatic amylase. We lack cellulase, so the beta-cellulose polymer fibers are eliminated in feces. Cellulose beta-structure provides considerable hydrogen bonding among its polar groups thereby conferring strength to the fibers for load bearing in plants and trees. [During polymerization, the C1 of the incoming glucose links to C4OH of the terminal (nonreducing) glucose of the existing polymer (or to C6OH, during branch creation); subsequent additions occur at both C4OH ends.] Each branch introduced doubles the substrate concentration of nonreducing ends to which new residues can be added. When 7-11 residues are added from the last branch, the outer four residues are moved to C6 of the 5th residue to create a branch. Once branched, the original molecule has 2, then 4, 8, 16, 32, 64, 128, 256, 512, 1024 termini after ten-branch events. If the glycogen molecule already had 1,000 linear chains, and each elongated and added 1024 branches, the concentration of substrate termini is over 1 million. (103 x 103 = 106). In sharp contrast, in starch, only one nonreducing C4OH end per polymer is available for addition or removal of residues. Thus, glycogen branching can accelerate glucose storage or debranching decelerates glucose release for mobilization from glycogen. Such exponential nonlinear growth helps bring postprandial hyperglycemia under control faster (glycemia), and provides for quick mobilization of glucose from glycogen during a heavy demand event (rapid exercise).

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Yes. In plants, glucose is released from stored starch during seedling growth (hours-days) before photosynthesis is available to synthesize glucose. Plant growth has no need for a quick (seconds-minutes) high demand addition/release polymer structure. The biochemical barrier that separates storage (starch, glycogen) from structural (cellulose) polymers is via a- and b-glucosides and their stereo specific enzymes. Note, after a meal, liver and skeletal muscle can store glucose into glycogen exponentially; conversely when sudden, high demand for glucose occurs, at the onset of vigorous excise, enormous quantities of glucose can be released from the considerable amount of glycogen macromolecular globules in these cells. [A short delay occurs during which stored ATP and creatine-P keep up with muscle demand for ATP until glycogenolysis, oxidative glycolysis, and mitochondrial respiratory oxidation take over ATP regeneration.]

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Of the five Rxns within the FA synthesis cycle, the 2nd and 4th use NADPH. The 2nd Rx reduces the β-keto –> β-OH, the 4th reduced the CH=CH to CH2-CH2. In the β-oxidation pathway, choice C then A. [1st Rx, FAD (to FADH2 introduces C=C, then 3rd Rx, NAD+ (to NADH converts b-CH2OH to C=O)]. The hexose monophosphate pathway (pentose shunt) produces NADPH in the 1st and 3rd dehydrogenase reactions. Deficiency of niacin, B3 or nicotinic acid [found in meats, nuts, legumes] leads to pellagra. Deficiency interferes with appropriate NAD(P)/NAD(P)H levels for FA synthesis, cholesterol, and complex lipids need for cell membranes and nerve insulation. Co-deficiency of riboflavin, B2 [same food source, pellagra] would interfere with FAD/FADH levels for various dehydrogenases and with β-oxidation (1st and 3rd reactions use FAD and NAD+, respectively). Hence, niacin deficiency interferes with lipid anabolism and catabolism. Glucose-6-phosphate dehydrogenase (G6PDH) deficiency leads to hemolytic anemia (see Clinical Case Presentation briefing sheet). Primaquine (and related antimalarials) undergoes redox reactions in the cell that produce large quantities of superoxide and H2O2. Superoxide dismutase converts superoxide into H2O2, which is inactivated by glutathione peroxidase that uses NADPH as coenzyme. If an individual has a genetic defect in G6P dehydrogenase (usually have an unstable enzyme with a shorter half-life in the RBC or an enzyme unusually sensitive to inhibition by NADPH. Hence, insufficient NADPH is produced by G6PDH resulting in impaired recycling of GSSG to GSH. Primaquine induced oxidative stress leads to lysis of RBCs (hemolysis) and hemolytic anemia. Patients may develop jaundice from released heme being catabolized into yellow bilirubin, and if severe enough, Hb appears in the urine, hematuria (dark-colored urine). RBCs lack DNA gene expression, so proteins/enzymes, including G6PDH, damaged by oxidative damage progressive accumulate leading to premature cell death when ATP production and cellular ion gradients can’t be maintained.

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Substrate availability in vivo is usually below the Km so that all the glycolytic reactions exhibit first order kinetics compared to zero order, which is usually never achieved except in vitro. Allosterism by three enzymes: hexokinase/glucokinase isozyme, PFK-1, and pyruvate kinase regulate glycolysis. Posttranslational covalent modification via protein kinase A phosphorylates (P) PFK-1 –> PFK-1P (inact.); similarly PK –> PK P (inact.) in liver.

[More depth. In muscle and most tissues, hexokinase (Km glucose 0.1 mM is much lower than blood glucose ~5 mM) limits glucose entry into glycolysis. In contrast, liver parenchymal cells contain glucokinase (Km ~10 mM, an isozyme of hexokinase, (hexokinase IV), its gene is inducible by high dietary glucose to increase glucokinase levels to meet demand. Hence, both liver enzymes enable it to “buffer” blood glucose levels when elevated glucose after a meal. Muscle hexokinase is product inhibited (G6P), which may help divert some glucose into replenishing or maintaining glycogen while glucose is also consumed glycolytically (if muscles are working lightly or moderately during and after a high glucose meal]. 81 Allosteric regulation of liver glucokinase is by (–F6P, +F1P, also binding of inhibitory protein) of PFK-1 (–ATP, +AMP, –citrate), and of pyruvate kinase (+F16BP, –ATP). 82 Glycolysis activity is stimulated by pancreatic insulin. It’s antagonist, pancreatic glucagon, inhibits glycolysis. Both are small protein hormones. 83 Both hormones. Insulin and glucagon bind to specific cell receptors which trigger dephosphorylation/phosphorylation, respectively of pyruvate kinase. Before breakfast, glycolysis is relatively inactive (PKp) because glucagon has been keeping the liver gluconeogenic. A significant portion of the liver glycogen has been depleted during the post absorptive state overnight. Muscle glycolysis is basal in the resting body and muscle proteolysis has supplied the liver with appropriate amino acids for gluconeogenesis keeping various active tissues (e.g., brain, heart and diaphragm muscles compared to other less active cells) supplied with glucose to sustain basal metabolic energy needs. After a glucose-rich meal (100 gm or more glucose) digestion and absorption floods the liver and body with a hyperglycemia spike. As soon as the pancreas β-cells detect the rise in blood sugar, stored insulin is released. Insulin stimulates receptors which leads to glycogenesis to replenish glycogen, glycolysis is activated (PK p dephosphorylated to active PK) and stimulated by 2,6BPF, fatty acid synthesis is stimulated) and the TCA rapidly replenishes bodily ATP levels after the cessation of the body’s resting period. After the absorbed glucose is depleted, the insulin levels drop after the second phase of insulin release, and as glycemia declines into early hypoglycemia, pancreas a-cells release glucagon to turn on glycogenolysis, slow glycolysis (PK to PKp inact), inhibit fatty acid synthesis. As glycogen depots decline, gluconeogenesis becomes activated again as is appropriate to maintain glycemia until the next meal. [note: any given cell may have large multiple sets of glycolytic pathway enzymes, at any given time. During which, a greater or lesser proportion of them may be at different activity levels, i.e., substrate, allosteric, modification.] 84

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A futile cycle consists of substrate A converted to product B by one pathway, which is converted back to substrate A by a different pathway, no net work accomplished, again and again. If ATP hydrolysis is involved, than the net effect of the futile cycle is the consumption of ATP. The metabolism of B doesn’t occur and ATP is consumed, not replaced. Once ATP is depleted, the cell, tissue, organ, or animal dies. To prevent futile cycles, one or both of the pathways is regulated and put into different cell compartments, e.g., fat synthesis and oxidation. Malonyl CoA (synthesized in the cytoplasm) is the activated form of acetyl CoA incorporation into fatty acids. Malonyl CoA also inhibits the carnitine acyl transferase (CAT) in the mitochondrial membrane, thereby preventing fatty acids from entering the mitochondria Inhibition of CAT by the substrate of fatty acid synthesis prevents reoxidation of newly synthesized fatty acids back to acetyl CoA; that prevents a futile cycle. Indirectly, malonyl CoA inhibits b-oxidation by cutting off the supply of its substrate: acyl CoA fatty acids. Malonyl CoA therefore controls both aspects of fatty acid metabolism. Acetal CoA in the mitochondria has no membrane transporter for it to leave the mitochondria. Thus, the flow of excess glucose carbons into fatty acids in the cytoplasm must enter as pyruvate but can’t leave the mitochondrion as acetyl CoA. Fatty acid synthesis in the cytoplasm is blocked because its substrate, acetyl CoA is trapped in the mitochondrion. Acetyl CoA condenses with oxaloacetate (OAA) via the first step of the TCA cycle forms citrate. Citrate easily leaves the mitochondrion via the TCA antiporter in exchange for a molecule of malate that enters into the mitochondrion. Mitochondrial citrate in the cytoplasm is cleaved to acetyl CoA and OAA by cytoplasmic citrate lyase which uses ATP and CoA as co-substrates. Breaking a C-C bond requires the considerable energy ATP hydrolysis provides, and enough also for the synthesis of the thioester of acetyl CoA: citrate + CoA + ATP --> OAA + acetyl CoA + ADP + Pi Acetyl CoA carboxylase is a biotin-dependent enzyme with distinct enzyme functions and a carrier protein function: biotin carboxylase, a transcarboxylase, and a biotin—carboxyl-carrier protein. The biotin carboxylase adds O=C=O to the C2 of acetyl CoA. That occurs in two stages: 1– carboxylation of biotin to biotin-N-COO– (ATP –> ADP + Pi), 2– followed by transfer of the carbonyl group to acetyl CoA –> malonyl CoA with release of the free enzyme—biotin complex. Beta oxidation of an odd-carbon fatty acid near its final stage yields propionyl CoA as the last fragment: CH3-CH2-CH2CH2-CO-CoA –> CH3-CH2-CO-CH2-CO-CoA (cleavage) –> CH3-CH2-CO-CoA (propionyl CoA) + CH3-CO-CoA (acetyl CoA). All three nucleoside drugs terminate chain elongation once incorporated into the chain because their structure lacks the group required for the next addition. (a) Azido thymidine (AZT)-5’-triphosphate contains the reactive –N=N+=N– on the

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3’ carbon (i.e., C3’–N3 carbon that replaces the –OH) of the deoxyribose component. (b) Dideoxyadenosine5’triphosphate contains 3’-H instead of –OH on the 3’ carbon of the deoxyribose component. (c) Puromycin resembles an the 3’ residue of a charged tyrosinyl-tRNA [the 3’ terminus adenosine of a charged tRNA containing tyrosine (i.e., dimethylaminoadenine-β-N-3’amino-(O-methyltyrosinyl) ribose] and mimics the acceptor role of the next amino charged tRNA into the A-site. Once the peptidyltransferse reaction transfers the nascent polypeptide to the puromycin, elongation ceases (the remainder of the tRNA structure is missing, the polypeptide-puromycin molecule is not bound to the ribosome). ATP-dependent kinases phosphorylate nucleosides to NMP, NDP, and NTP nucleotides, phosphatases remove the phosphates. Nucleoside transglycosylases exchange one base for another. Salvage enzymes directly convert free bases to the nucleoside-monophosphate nucleotide in one step using PRPP as phosphoribosyl donor. AZT deoxynucleoside is phosphorylated to 5’dAzTTP and mimics 5’dTTP. Lacking phenylalanine hydroxylase, phenylalanine cannot be hydroxylated to tyrosine in patients with PKU, hence the corresponding deaminated keto acid, phenylpyruvate, and its reduced analog, phenyllactate, are elevated in the urine of untreated patients. If PKU is undetected in newborns, severe mental retardation develops. A drop of blood is taken from the heel of all newborns in the US (required by law) for required routine phenylalanine analysis. The mental retardation of PKU is preventable, if detected soon after birth. Poorly educated immigrants from remote areas of the world may be ignorant of this inborn error of metabolism, they, their developing unborn children, and infants are sensitive to the toxic effects of high concentrations of phenylalanine and related phenylketones. A diet restricted in phenylalanine but supplemented with tyrosine prevents the mental retardation and neuropathology from developing. Too little phenylalanine (phe) in the diet can cause negative nitrogen balance accompanied by lower protein synthesis in body tissues, with wide ranging consequences. Too much dietary phe is associated with neurotoxicity effects. Supplementary tyrosine can lessen it’s the risk of deficiency (phe is an essential amino acid by definition for PKU), by bypassing the missing enzyme step that makes tyrosine non essential in the normal population.. Although milk caries could account for the rotting teeth, and the very light pigmentation might suggest albinism due to a defect or lack of tryrosinase and the two step process involving the hydroxylation of tyrosine to dihyroxyphenylalanine (DOPA) and subsequent oxidation to a quinone, which leads to the formation of melanin in melanocytes. Neither situation is germane, but the mental retardation is. The unusual gait, sitting posture, and family history of epilepsy are telling. Interestingly, very light pigmentation may accompany PKU. Occasional unintended phenylalanine dietary excess can be treated with tyrosine supplementation to decrease the increased phe:tyr ratio to the more normal range, thereby making phe relatively less pronounced.

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Heme synthesis begins and ends in the mitochondrion (Baynes Fig. 27.4). Heme is part of cytochromes of the electron transport system (ETS). Synthesis begins with glycine and succinyl CoA (TCA intermediate) yielding 5-ALA via ALA synthase (heme is allosteric inhibitor). Two 5-ALA molecules exit to the cytoplasm where they condense into a pyrrole (5-membered N-ring compound). Four pyrroles assemble into a porphyrin in the cytoplasm. Side-chain modifications lead to uroporphyrinogen III, then to coporphyrinogen III, which reenters the mitochondrion. Additional side-chain modification yields protoporphyrinogen IX, then protoporphyrin IX. Ferrochelatase adds Fe2+ to yield heme. Genetic defects in the pathway are associated with various Porphyrias depending upon which synthetic enzyme step is deficient. 101 The synthesis begins and ends in the mitochondrion, with precursor porphyrins formed in the cytoplasm that enter the mitochondrion for final reaction steps. Heme is catabolized to bilirubin (yellow) in the liver. About 75% is derived from hemoglobin of senescent red blood cells which are phagocytized by (1) mononuclear cells of the spleen, (2) bone marrow, and (3) liver, which yields a daily load of 250-350 mg bilirubin. 102 Jaundice is a yellow color imparted to the skin and eye whites due to excess bilirubin (more than 50 µmol/L or 3 mg/dL in the plasma) often due to enlarged liver (hepatomegaly), which can have various etiologies, e.g., chemical (alcohol) and viral (hepatitis virus), and others. In β-globin, Glu6 (normal hemoglobin A (HbA) –> Val6 in HbS involves an A –> T substitution in the DNA (GAG –> GTG). 104 Under conditions of low oxygen tension (low pO2) or hypoxia, the single amino acid nonconservative mutation (i.e., amino acid change has different physical and chemical properties than the original) change (acidic for a nonpolar, hydrophobic side-chain) causes the HbS molecule to change shape that leads to HbS polymerization into rod-shaped structures that deform and alter the rheological properties of red blood cells (Baynes Fig 31.3). 105 Intermittent episodes of hemolysis and especially vaso-occlusive crises lead to severe pain in bones, chest, and abdomen. Also likely is an increased susceptibility to infections and multiple organ damage. 103

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Some enzymes are synthesized inactive, in a precursor form (proenzyme or zymogen). When needed, zymogens are activated, usually by proteolysis so the protein conformation changes to the active enzyme . 107 If an enzyme can catalyze the appearance of a second active enzyme by 10,000-fold, and it can catalyze the appearance of another active enzyme by 10,000-fold, the rate of the reaction of the last enzyme increases by ten million-fold! For certain processes, extremely short duration activation is essential if the response is to counter a sudden time-dependent, life threatening event such as loss of blood by trauma. Serial zymogen activations are referred to as zymogen cascades. 108 Blood coagulation has two serial zymogen cascades that have different zymogens but activate the same final zymogen, prothrombin. The intrinsic pathway circulates all the zymogens in the blood; the a second extrinsic pathway (outside of the blood) is triggered when cells membranes are ruptured at the site of physical trauma. In a different zymogen cascade, signal transduction rapidly triggers cascade-like intracellular events after receptor binding of specific ligand, e.g., insulin, glucagon, epinephrine, cortisol and other hormones. 109

The signal peptide is composed of predominantly hydrophobic amino acids (ala, val, leu, ile, phe, met) and is located at the N-terminus of certain proteins. 110 Only proteins excreted by the cell contain the signal peptide. 111 Proteins inside the cell are intracellular, those outside the cell are extracellular. Proteins synthesized by the cell that remain in the cell are endogenous proteins. Exogenous proteins made outside the cell they enter. 112 The Signal Recognition Particle (SRP) complex binds the signal peptide emerging from the ribosome that is actively translating the mRNA coding for an extracellular protein (e.g., collagen, fibrinogen, plasma fibronectin, immunoglobins, etc) thereby the nascent polypeptide is identified and sorted from the other mRNAs coding for endogenous proteins (e.g., metabolic pathway enzymes, tubulin, etc). 113 The SRP-mRNA-ribosome complex binds to the SRP docking complex on the endoplasmic reticulum membrane. The signal peptide is fed through the ER into the lumen where the signal peptidase removes the signal peptide (later digested to amino acids). Removal of the signal peptide is a posttranslational modification. The ribosome completes translating the mRNA coding region and the polypeptide chain ends up within the lumen where it is undergoes additional posttranslational modification in the Golgi apparatus. [Described in the Cell Organelles course.] 114

A nucleic acid consensus sequence is composed of nearly the same sequence bases found in different DNAs. E.g., TATAA box in promoters, homeobox in homeotic genes, etc.

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The promoter upstream TATA box sequence is bound by a protein factor that assists the RNA polymerase align at the 1+ nucleotide of the gene transcription region during the initiation stage of RNA synthesis. Not all genes have a TATA sequence, the lac operon lacks a TATA box, for example. 116 For mRNA synthesis, TATA (Pribnow box at –10 bp in prokaryotes bound by sigma factor, Hogness box at –35 in eukaryotes bound by the TBP), CAAT box (about –80 bp, bound by NF-1/CTF protein), GC box (bound by SP-1 protein). Enhancers and response elements are often within 1 kbp of +1 start-nt. For processing, consensus splice sites --AG/GU-intron-AG/GN--, AAUAAA signals poly A tail addition. For translation in prokaryotes, purine-rich ShineDalgarno AGGAGGU sequence is about 10 bp upstream from first 5’AUG and aligns with 3’UCCUCCA5’ in 16S rRNA; in eukaryotes, 5’cap, m7GpppN—, is bound by eIF-2. 117 Capping protects the 5’mRNA terminus from exonucleases. The m7GpppN— cap has (+) charge recognized by eIF-2, uncapped (pppN-mRNA) or GpppN-capped mRNA have only about 1% translation efficiency of m7GpppN-cappedmRNA. The 3’ poly A tail is added to only a few percent of HnRNA primary transcripts synthesized. The poly A likely assists in transport of mature mRNA from nucleus into cytoplasm for translation into protein. 118

Prokaryotic mRNA has 5’pppN-terminated mRNA with Shine-Dalgarno (S-D) consensus sequence near first 5’AUG. Eukaryotic mRNA has 5’m7GpppN cap (with + charge, bound by eIF-2) translation begins at first 5’AUG. 119 The distance between the S-D sequence upstream from the AUG start codon ensures the correct alignment of the prokaryotic ribosomal subunit P-site at the first 5’AUG due to complementarity the 16S rRNA in the smaller subunit. 120 For a second or additional cistron in a polycistronic mRNA, each cistron has a S-D sequence upstream of the initiation AUG of that cistron’s coding region. Hence, each internal cistron can be translated correctly beginning at the corresponding N-terminal f-met amino acid sequence. 121 The 30S ribosomal subunit would not be able to locate the correct 5’AUG at the beginning of the coding region for the affected internal cistron proteins and they would likely not be synthesized. Coordinated synthesis of the multigene operon proteins would fail. Would this likely be found in viable cells? [Not likely]. 122 123

The transcription of DNA into RNA in eukaryotes, capping does not occur in prokaryotic RNA transcription. A modification of the polynucleotide after its synthesis is completed by the DNA-dependent RNA polymerase. The exception is the 5’capping and cap methylation(s). These occur on the nascent RNA transcript shortly after transcription has begun (i.e., long before transcription is complete). Posttranscriptional modifications include any removal of sequences

from mRNA, tRNA, and rRNA primary transcripts (these are longer than their mature and functional molecules), methylations, splicing, polyadenylation, and other changes to the sugar or base of specific nucleotides in the RNA. 124 Capping transfers pG (GMP) of pppG (GTP) to the 5’ppN-terminus of the pre-mRNA (HnRNA, in the nucleus) yielding the 5’capped mRNA (GpppN-terminus). Subsequently it is methylated using SAM to yield cap: m7GpppN-. Additional base or ribose methylations can occur, m7GpppNm-, m7GpppmNm- depending upon which gene’s mRNA is being synthesized. 125

Eukaryotic initiation factor eIF-2. [Oocyte maternal mRNA have GpppN-terminated mRNAs and these are not translated until fertilization after with the caps are methylated and are then translated for the first embryonic developmental sequences to begin.] 126 Coenzyme NAD: nicotinamide(5’)ppA, NADP: nicotinamide (5’)ppAp; FAD: flavin-ribitol(5’)ppA resemble mRNA caps: m7G(5’)pppN (where N = A,G 95%). 127

All act on nucleic acids. dsDNA is acted on by restriction enzymes (site-specific cleavage), topoisomerase (unwind, and wind a-helix, winding is ATP-dependent), ligase (repairs single-strand nicks) ssDNA exo- and endonuclease (hydrolyze nt at end or within the strand, respectively) and polymerases (most are template-directed assembly of linear DNA or RNA). RNA viral-encoded enzymes act on dsRNA and ssRNA dependent upon the biological entity under consideration. Reverse transcriptase copies RNA into complementary ssDNA then copies the ssDNA into dsDNA (e.g., lysogenic tumor viruses).

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Restriction enzymes restrict (i.e., prevent) exogenous genetic nucleic acid (usually DNA) from expressing its biological potential in prokaryotic cells. A kind of enzyme-based anti-DNA defense system. 129 The cell’s DNA is methylated at the specific sequences recognized and cleaved by the restriction enzyme(s) in the cell. Incoming exogenous DNA that lacks site methylation protection is restricted by the cellular enzyme thereby fragmenting the DNA making it unviable (especially if the cleavage(s) occur in foreign genes. 130 If a piece of DNA has one or more restriction sites and is treated with one or the other corresponding restriction enzymes, the lengths of the DNA produced will be unique for each restriction enzyme digest and equivalent to a (length) fingerprint. For medical detection of pathologic mutations, the sickle cell anemia mutation (Glu 6 GAG  Val6 GTG) can be screened in a patient’s DNA because the mutation abolishes a recognition site for Mst II (CCTN(A–>T)GG). The CCTNAGG in HbA gene (corresponding to Glu6) is cleaved but the CCTNTGG in the HbS gene (Val6) is not cleaved. Thus, patients with sickle cell anemia will show only one band (1.4kb), while carriers will have 2 bands (one 1.4 kb and another 1.2 kb, and unaffected individuals will have a single 1.2 kb band (see Baynes Fig 33.17.) 131

DNA restriction enzymes cleave at specific tetramer or hexamer bp sequences that are palindromic (same sequence on both antiparallel strands). Methylation (once or twice) protects the site from cleavage. Thus the cell methylates these sequences preventing destructive hydrolysis and fragmentation of its own DNA. 132 Some restriction enzymes cut straight through both strands at a specific bp yielding fragments with blunt-(flush)-ends (e.g., Hae III, EcoRV, Bal I); others restriction enzymes stagger the cut of each strand to yield sticky ends (self-adhesive or annealing ends, e.g., EcoRI, Msp I) [see Baynes Fig 33.8]. 133 The DNA ligase works by a two-step mechanism. (a) It activates the phosphate at the nick, pN—by adding AMP, i.e., Ap-pN---intermediate (i.e., mimicking the incoming dNTP, pppN in the polymerase reaction). (b) The NOH3’ at the nick displaces the AMP forming the –N—p-N- phosphodiester bond, the same outcome as the DNA polymerase reaction. The ligase is not template directed, it doesn’t replace any nucleotides, it accepts whatever 5’pN-- is opposite the NOH3’, and merely repairs a broken O—P bond. 134 DNA topoisomerases, like DNA ligases can make a O—P bonds to complete the phosphodiester linkage of the backbone. However, topoisomerases first broke that bond, wind (ATP-dependent) or unwind the DNA one turn (topo I), or two turns (topo II), before repairing the same O—P breaks. 135 Polynucleotide phosphorylase (PNP) reversibly polymerizes 5’ribonucleoside di-phosphates (i.e., nucleoside pyrophosphates) to polynucleotides according to: n NDP --> (Np)n + n Pi. DNA-directed RNA polymerase reversibly polymerizes 5’ribonucleoside tri-phosphates to polynucleotides according to: n NTP + DNAtemplate--> (pppN(Np)n-1 + n Pi. The PNP differs from DNA template-directed RNA polymerases because it lengthens an RNA primer to generate a random sequence whose composition (ratio of bases) is dependent upon the substrate ratio of ADP, CDP, GDP, and UDP in the reaction mixture. RNA polymerase generates a nucleotide sequence complementary to the template, it needs no primer. 136 A plasmid is a self-replicating extrachromosomal DNA found in some bacteria. The number of copies the plasmid reproduces of itself in the bacterium carrying it is the copy number, it is dependent upon the plasmid examined and may range from none to almost two dozen. Some plasmids carry genes coding for enzymes that inactivate particular antibiotics (ampicillin, tetracycline, are examples) thus providing the bacterial cell with protection against the antibiotic.

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These ampicillin (like penicillin) and tetracycline resistance genes were experimentally incorporated into the pBR322 plasmid in the laboratory for use in monitoring the steps involved in the molecular cloning of a piece of DNA. Within the ampicillin resistance gene is a Pst I restriction site, and within the tetracycline resistance gene is a Sal I site; between these genes is an Eco RI site. Insertion at the Eco RI site has no effect on both drug resistances. Insertion of DNA into the Pst I site inactivates the ampicillin gene (insertional inactivation). Cells containing this recombinant (passenger DNA in the plasmid vehicle) are killed by ampicillin but are still resistant to tetracycline, for example. 138 In practice, the plasmid preparation is a mixture of original plasmid molecules with drug resistance and new recombinant plasmid molecules with inactivated resistance genes containing the inserted DNA. At a multiplicity of infection (MOI) such that each cells receives, on average, only one plasmid. Cells receiving no plasmid or the recombinant die, while cells receiving the original plasmid survive exposure of the drug. 139 Two problems occur: was the insertion successful, and did the plasmid get into the test cells? Insertion of a DNA into the EcoRI site between the ampicillin and tetracycline genes of pBR322 may not be successful, but how does one know. By using insertional inactivation at the Pst I site in the ampicillin gene, the DNA is inserted, the cells with the inactive ampicillin gene die when given ampicillin. Thus, the recombination is verified for the passenger DNA insert. The tetracycline gene is still active. Tetracycline resistance verifies that the plasmid was successfully introduced into the host bacterium (transfection) because the cells survive treatment by tetracycline. Knowing the transfected cells contain the appropriate recombinant, the molecular cloning can proceed. Thus, one could produce large quantities of insulin DNA by growing a large volume of transfected cells. 140

The open-ring form of glucose exposes the highly reactive C1-aldehyde that can react with exposed amino groups. Blood distributes glucose continuously throughout the body. The rate of protein glycosylation by glucose is concentrationdependent. The mole fraction of the N-terminals of the β-chains of hemoglobin A in red cells derivatized by glucose is used as a measure of blood glucose level during previous weeks. The 120-day life span of the RBC provides a backward assessment of accumulated glycosylation that reveals quantitatively the patient’s management of (a) dietary glucose load and (b) how the body has managed glucose metabolism. Normal metabolic control of glycemia gives up to 6% glycosylation (HbA1c). Increasing dietary glucose intake prolongs increases the net duration and severity of hyperglycemia spikes and the HbA1c values are progressively above 6%. For diabetics, this provides an objective measure of the patient’s history independent of personal memory of diet and medication practices for at least 3 months. 141 Impaired insulin secretion and/or effectiveness (insulin resistance due to prolonged high dietary glucose intake) correlates with faulty glucose homeostasis and utilization of fatty acids as fuel by body tissue cells that is also associated with polyphagia (excess appetite) accompanied by decreasing body weight. The body perceives starvation, polyphagia inappropriately ingests more nutrients even though sufficient dietary nutrients are available within the body. 142 Type 1 and 2 DM differ in early v. later age onset; abrupt vs. gradual; normal v. obese weight; HLA associated as positive v. negative; little or no blood insulin v. some-normal-high; islet cell antibodies present at onset v. absent; islet antibodies present at diagnosis v. no; insulin synthesis is absent immune destruction of b-cells v. combination of impaired b-cell function and insulin; prevalence is 0.2- 0.3% v. 2-4%; polyuria, polydipsia, polyphagia, weight loss, ketoacidosis v. polyuria, pruritis (itching), peripheral neuropathology, ketoacidosis after major stress; 143 Animal protein is recognized as non-self by the immune system, which produces antibodies that counter the effectiveness of exogenous animal insulin treatment. Exogenous human insulin is non antigenic. 144 The molecularly cloned insulin can be obtained in highly pure form and is not antigenic. 145

Actinomycin D at different doses differentially inhibits DNA and RNA polymerase. Methotrexate inhibits folate reductase. Tetracycline and puromycin inhibit elongation and initiation of protein synthesis, respectively. Trimethoprim inhibits bacterial met-tRNAimet transformylase. 146 Methotrexate is a folate analog that inhibits folate reductase, which is part of the three enzymes concerned with dTMP synthesis from dUMP. The folate inhibitors effectively stop dTMP synthesis. More metabolically active cancer cells are killed than normal body tissue cells. After a calibrated period of treatment, further inhibition is stopped by injecting folate to out compete the drug, i.e., significantly increase the folate:methotrexate ratio in cells. The most active cells of the body are more severely affected then less metabolically active cells accounting for the side affects of chemotherapy. 147 See footnote 145. 148 GI tract epithelia have a fast turnover rate and active hair follicle cells are particularly affected leading to GI malaise and hair loss, respectively. 149 In prokaryotes: tetracycline and trimethoprim; in eukaryotes: methotrexate; in both cells: actinomycin D and puromycin.

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Cobalamine is both vitamin and active coenzyme. The others require an additional component for activity: thiamine and pyridoxine have a pyrophosphate and a phosphate added, respectively, as coenzyme; riboflavin is combined with AMP and niacin is combined with ribosyl-ADP before active as coenzymes.

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Oxidation/reductions: FAD and NAD dehydrogenases; 1-carbon transfers: folate (not listed, e.g., N10-formyl-FH2), 2carbon transfers: coenzyme A (not listed, e.g., acetyl CoA is used for N-terminal acetylation, e.g., eye lens α-crystallin protein). 152 All listed vitamins are water soluble, also vitamin C, folate, pantothenate, biotin. Fat-soluble vitamins are A, D, E, and K. 153 Thiamine (B1): major signs and symptoms is peripheral neuropathy, loss of appetite, constipation and nausea occur early, then depression and confusion. Impaired nerve cell function leads to beriberi characterized primarily by advanced neuro-muscular symptoms. Riboflavin: inflammation of the corners of the mouth (angular stomatitis), tongue (inflammation of glossitis) and scaly dermatitis. Niacin: dermatitis, diarrhea and dementia in severe deficiency in pellagra (rarely seen in the modern world). Pyridoxine: peripheral neuropathy, convulsions, coma in later severe form, early onset shows irritability, nervousness and depression in mild form. Cobalamin: megaloblastic anemia also involves folate (methylmalonic aciduria and homocystinuria) and pernicious anemia (due to lack of intrinsic factor protein (IF) in the stomach and hence absorption of B12 is prevented). 154

Pyridoxine is the major form in the diet, and pyridoxal phosphate (via pyridoxal kinase) is the active form of the vitamin. All nonphosphorylated and phosphorylated forms are water soluble. 155 Pyridoxal-P forms a Schiff base intermediate with the α-NH2 of amino acids. One of three bonds can then break yielding (a) amination/deamination, (b) loss of carboxyl group, or (c) dehydration (serine) of the amino acid. 156 Amination converts their α-keto forms to amino acids (e.g., pyruvate to ala, OAA to asp, a-KG to glu) for amino acid synthesis via aminases/transaminases. Deamination to the α-keto forms is the first step in catabolism. Either is used to interconvert α-keto and α-amino acid forms. 157

Iron in RBC hemoglobin, muscle myoglobin, cytochromes and iron-sulfur complexes in mitochondria are important for oxygen transport and energy metabolism. Copper in several oxygenases, these include cytochrome c oxidase, and several cuproenzymes involved in heme synthesis, superoxide dismutase, and ceruloplasmin synthesis and function. 158 Iron anemia. 159 Ceruloplasmin in plasma scavenges superoxide and other oxygen free radicals. Ceruloplasmin is defective in Wilson’s disease. [Ceruloplasmin produces a sky blue color in solution similar to cerulean blue pigment. Recall solutions of copper ions are blue such as copper sulfate.] 160 Ferrous iron is stored by ferritin protein in most body cells, ferric iron is transported by transferrin protein. Ceruloplasmin is the major transport protein of copper to the body and is also essential for the regulation of the oxidationreduction reactions, transport, and utilization of iron (see Baynes Fig 3.6). That is, ferritin-Fe2+ + ceruloplasmin-Cu2+ <–> transferritin-Fe3+ + ceruloplasmin-Cu+. Ferritin scavenges free iron (generally toxic, especially in the central nervous system). Oxygen or oxidized thiol groups reduce Cu+ to Cu2+ in ceruloplasmin. Hence, if copper is deficient, iron metabolism is effected. Functional hemoglobin and myoglobin contain Fe2+. Occasionally it is oxidized to Fe3+ when bound O2 is reduced to superoxide nonenzymatically to yield ferrihemoglobin or methemoglobin (rust brown color), which binds oxygen poorly. Methemoglobin reductase restores the Hb(Fe2+). [in patients with metHb reductase deficiency (methhemoglobinemia) have a dark cyanotic appearance; vitamin C treatment reduces the iron back to ferrous Fe2+..] Copper deficiency also is associated with degeneration of vascular tissue with bleeding due to defects in elastin and collagen production. Since copper deficiency influences iron metabolism, small copper deficiency manifests as microcytic (small erythrocytes) microchromic (pale erythrocytes) anemia that is resistant to iron therapy. 161

All 20 amino acids are need to synthesize about 200 g total protein per day in the healthy 70 kg human. The body can synthesize 12 amino acids by metabolic pathways, but eight amino acids cannot, those eight are: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine, leucine, lysine (memory acronym: Pvt. Tim Hall). Their carbon skeletons cannot be synthesized by the body thus the diet becomes essential in supplying them in an adequate amount based upon their composite average mole fraction of daily protein synthesis. 162 Nitrogen balance occurs when the dietary intake of utilizable nitrogen equals the nitrogen excreted (sweat, urine, feces). Body composition of nitrogen is mostly accounted for by protein mass (about 16% N), the N in nucleic acids and some other metabolites is ignored. 163 For infants, human milk is the best source because the ratio or mole fractions of the amino acids matches the amino acids comprising the total body proteins of the infant. Moreover, human milk protein is completely digestible and absorbable for full utilization. Bovine milk is ideal for calves for the same reason and is the next best source for humans, hence its wide commercial availability. Eggs are the next best source of protein, the high cholesterol content notwithstanding. [Beside the lactose intolerance, a portion of the population has allergic responses to cows milk.]. 164 As an essential amino acid, the body can’t synthesize it to maintain its representation in the daily requirement for protein synthesis needs. All proteins initiate with methionine, so a 50% decrease in availability could decrease protein synthesis by the same amount. Additionally, all other proteins will be affected in proportion to the number of internal methionines.

Once started, polypeptide elongation halts at internal methionine codons in proportion to the decreased methionine concentration, in this case 50%, thus, the overall rate of elongation is decreased by at least 50%, perhaps more in selected cases. 165 While essential amino acid deficiency is ongoing, the turnover of proteins will release some of the deficient amino acid(s) back into the amino acid pool (cellular, tissue, organ, whole body) and that helps de novo protein synthesis, but the nonessential amino acids can’t be incorporated because overall protein synthesis is about 50% (met example). The nonessential amino acids are processed catabolically through oxidation which begins with deamination. The deaminated N is incorporated into urea to prevent ammonia toxicity. The net result is the loss of body N now exceeds diet intake, hence, negative nitrogen balance ensues and continues until the deficiency of essential amino acids ceases. 166

See Baynes Fig 28.7. (a) Ribose reduction, all NDP + thioredoxin-(SH)2 (NADPH) via ribonucleotide reductase --> dNDP + thioredoxin-S2 (NADP+). (b) Uracil to thymine, dUMP + N5N10-methylene-FH4 via thymidylate synthase --> dTMP + dihydrofolate (FH2). 167 dUMP to dTMP (see fn 167). ancillary Rx 1: FH2 + NADPH via FH2 reductase --> FH4 + NADP+. Rx 2: FH4 + serine via serine hydroxymethyl transferase --> N5N10-methylene-FH4 + glycine + H2O. 168 The ribonucleotide reductase uses NADPH which has niacin (B3, nicotinic acid). The dihydrofolate reductase FH4 uses folate [also multiple peptide composed of glutamate]. 169 A hydride is a proton with two electrons or H:–, the hydride equivalent is donated by the two cysteine –SH groups that form a bridged thioredoxin–S-S-intrapeptide. These are reduced in turn by NADPH. Nicotinic acid is the vitamin and its amidation yields the active nicotinamide unit of NADPH. [note: During deoxyribose formation, the two –SH groups in thioredoxin (–trp-Cys-gly-pro-Cys-) donate a hydride equivalent (H–) and are oxidized to cross linked cystine disulfide. Reduction back to a pair of cysteines (–trp-Cys-gly-pro-Cys-) is via flavoprotein thioredoxin reductase. NADPH (from pentose shunt) as electron source reduces the –S-S- back to active thioredoxin–(SH) 2 . In the second reaction (reduction of FH2 to FH4), NADPH donates the hydride + H+ (i.e., H2) hydrogenation equivalent.] 170 In addition to tetrahydrofolate transferring carbon atoms (in the forms of formaldehyde, formyl, hydroxymethyl, methyl, or forminimo), S-adenosyl methionine (SAM) for methylations, and CO2-biotin for carboxylations. 171

There are over 300 known zinc-containing enzymes. Aldolase, alcohol dehydrogenase. Metallothioneines are small proteins containing about 62 amino acids with about 30 mole % as cysteine. These provide pockets of S-donor atoms that can bind heavy metals, especially Cu2+ and Zn2+. Cadmium induces the cellular resistance to heavy metal cytotoxicity by stimulating thionein synthesis, which is mediated in most species by the binding of metal ions either to a cysteine-rich polypeptide in the metallothioneine family or to short cysteine-containing gamma-glutamyl peptides. The thioneines are important in heavy metal detoxification and excretion. 173 For metalloenzymes, deficiency of the metal cofactor inhibits enzyme activity because the chemistry of the catalysis involves the metal ion. In Wilson’s disease, the liver’s capacity to synthesize ceruloplasmin is impaired leading to chronic accumulation of copper in body tissues associated with hemolysis and damage to both liver (cirrhosis) and brain cells. The activities of copper, zinc, iron, and other metalloenzymes will be affected by the excessive copper accumulation, which can interfere with absorption and utilization of other metal ions. 172

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Vit D: bones, curved legs. Vit C: subcutaneous and other hemorrhages, muscle weakness, soft, swollen bleeding gums, osteoporosis, and poor wound healing and anemia. Niacin: dermatitis, diarrhea, and dementia. Thiamine: beri-beri, early loss of appetite, constipation, and nausea, later depression, peripheral neuropathy and instability and impaired nerve function with advanced neuromuscular symptoms. Folate: megaloblastic anemia due to failure to synthesize nucleic acids (folate-dependent, 1-carbon metabolism). Polycythemia (erythrocytosis) is an increase in the hematocrit 175 Water soluble: vit C, niacin (B3), thiamine (B1), and folate are water soluble [also flavin (B2), pantothenic acid (B5), pyridoxine (B6), biotin, cobalamin (B12)]. Lipid or fat soluble: Vit D [also vit A, E, K]. 176 Vit D (rickets): fish oils and egg yolks. Vit C (scurvy): citrus fruits. Niacin (the 3 Ds): meats, nuts, legumes. Thiamine (beri-beri): seeds, nuts, wheat germs, legumes, lean meat. Folate (anemia): yeast, liver, leafy vegetables. 177

After dTMP synthesis, dihydrofolate reductase converts FH2 to FH4 (tetrahydrofolate). DHF reductase is inhibited by folate antagonists such as methotrexate (also related drug aminopterin). If FH4 cannot be regenerated, the 1-carbon-FH4 derivatives pool becomes used up, 1-carbon metabolism ceases, the cell dies. Thus, methotrexate blocks FH 4, regeneration, the next ancillary reaction cannot regenerate N5N10-methylene-FH4 from serine and continued dUMP to dTMP synthesis ceases. 178 Pentose shunt. First reaction via glucose-6P-dehydrogenase and 3rd dehydrogenase (an oxidative decarboxylation) regenerate NADP+ to NADPH, which supplies the H2 equivalents to FH2 reductase to regenerate FH2 to FH4. 179 The 3rd carbon (the hydroxymethyl side chain –CH2OH) of serine, is transferred to FH4 onto N5N10. 180 The dUMP to dTMP reaction produces FH2 (dihydrofolate), FH2 has no further metabolic use.

8888 Endnotes editing stopped here 8888 181

ECM is a complex network of secreted macromolecules located in the extracellular space. The EMC of skin and bone provides the structural framework of the body. In all tissues the EMC has a central role in regulating basic cellular processes, including proliferation, differentiation, migration and cell-cell interactions. The EMC macromolecular network is composed of collagens, elastin, glycoproteins and proteoglycans, secreted by connective tissue cells such as fibroblasts and epithelial cells. 182 The total body mass is about 25% collagen and collagens are the primary structural components of the EMC in connective tissues. Collagen’s three polypeptide chains [19 different collagen types, composed of 34 related, but distinct polypeptide chains] of 600 to 3000 residues are wound about each other in an extended α-helix conformation found in globular proteins. Glycine has the smallest side-chain (H), therefore the Gly-X-Y tripeptide allows the triple helical stands to fit closer together. The X and Y (most often pro and hydroxy-pro) confer rigidity to the molecule because of their bulk and limited rotation about the α-amino group. Intra- and interchain helices are stabilized by hydrogen bonds mostly between NH and C=O groups. The side chains of the X and Y groups point outwards from the helix where they can form lateral interactions with other triple helices or proteins (4º) stated in the correct answers of the test question. 183 The long cylindrical-shaped collagen molecules align in parallel quarter-staggered arrays (like bricks in a wall) such that the ends overlap and appear as bands across the fibers (resemble fibrin fiber staggered-arrays) when viewed in the electron microscope. 184

Glucose, mannose, galactose, ribose are simple sugars. Glucosamine, mannosamine, galactosamine, found in complex carbohydrates, and deoxyribose are derived sugars. Ribose is in RNA, deoxyribose is in DNA. 185 Glycoconjugates are formed usually with glucuronic acid to detoxify molecules that have a carboxyl or hydroxyl group. Enzymes conjugate either through an ester or an ether type of linkage. Glucuronic acid conjugates with bilirubin. [Another conjugate: glycine + benzoic acid gives hippuric acid, a compound used to measure liver function. Glucuronic acid HOOC-(CHOH)4-CHO is formed from glucose by oxidation of the 1° alcohol of the last carbon, i.e., C6-OH.] Glycolipids are classified into four groups: cerebrosides, sulfatides, globosides, and gangliosides. In all these classes, the polar head-group – comprising the sugars – is attached to ceramide by a glycosidic bond at the terminal OH of sphingosine. 186 Proteoglycans are gel forming components of the ECM. Those listed are glycosaminoglycans (GAGs) composed of repeated disaccharides: hyaluronic acid (GlcNAc and GlcUA) and is the longest of the GAGs (250-25,000 residues); heparin (IdUA and GlcNAc); chondroitin sulfates (GlcUA and GalNAc), dermatan sulfates (IdUA and GalNAc), heparin sulfates (IdUA and GlcNAc), and keratin sulfates (Gal and GlcNAc) have sulfate on some of their amino groups. 187

There are seven crystal systems: cubic, tetragonal, orthorhombic or rhombic, monocline, triclinic, hexagonal, rhombohedral or trigonal. Calcium phosphate is in the hexagonal system (four axes: three equal coplanar axes at 60°, and one at right angles to them) and is known as hydroxyapatite found in bone and enamel. 188 Enamel hydroxyapatite is the most calcified and hardest mineral in the body. In decreasing hardness is enamel > dentine ~ cemented > bone. Mineralogists us the Mohs Scale of Hardness: from softest is 1. talc 2. gypsum, 3. calcite, 4. fluorite, 5 apatite, 6. feldspar, 7. quartz, 8. topaz, 9. corundum or sapphire, to hardest 10. diamond. 189 Amelogenins are highly conserved 7-25 Kd proteins unique to enamel; they compose about 90% of developing enamel ECM concerned with enamel mineralization and are high in proline and glutamate. Excreted by the ameloblasts, the amelogenins are bipolar, nonglycosylated proteins with some phosphoserine residues; the genes are on both human X and Y genes. DNA sequences for human X and Y genes, bovine X and Y genes, opossum and mouse genes are known. Amelogenins self-assemble into nanospheres (10-20 µm diameter) of about 100 monomer units, with the C-terminals on the surface. Initially they bind to the protoenamel on the a- and b-faces allowing crystal growth only on the c-face surface; as crystallization proceeds, during maturation, the amelogenins are lost allowing growth on the a- and b-faces. 190 Exchange of F– for OH– yields fluoroapatite, which is harder than hydroxyapatite. 191

Diacylphosphoglycerol (DAG) is glycerol esterified to two fatty acids; esterification of the end OH of DAG with phosphate yields a phospholipid. Additional esterification the phosphate by choline, serine, ethanolamine, or inositol yields the corresponding phosphatidyl choline, etc. All these are found in cellular membranes. 192 Ethanolamine is decarboxylated serine. Choline is derived from ethanolamine by three N-methylations (SAM) to yield a quaternary nitrogen with a positive charge. Betaines are amino acids with fully methylated amino groups. Thus, the simplest betaine (of glycine) is derived from choline by two oxidization steps: –OH to –CHO aldehyde then to –COOH yielding glycine betaine or betaine: (CH3)3N+–CH2–COOH. Methionine is salvaged from betaine + homocysteine by a transmethylase which also yields dimethylglycine (two additional demethylations yield sarcosine then glycine). 193 Phosphatidyl lipids comprise the major amphipathic molecules of the structural portion of cellular membranes.

194

The glutamate decarboxylase produces GABA. Ornithine decarboxylase yields putrescine, tryptophan decarboxylase yields tryptamine, glycine decarboxylase yields taurine, and aspartate decarboxylase yields β-alanine (constituent of pantothenate) [Also important are lysine to cadaverine; tyrosine to tyramine; histidine to histamine.]. The vitamin is B6 (pyridoxine) is active as pyridoxine phosphate in the decarboxylases that use pyridoxine phosphate. 196 GABA is the abbreviation for γ-amino-butyric acid derived from glutamine. 197 GABA is the major inhibitory neurotransmitter in brain tissue. 195

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Myoglobin is a 16.9 kd single polypeptide containing heme in muscle; hemoglobin is a 63 kd tetramer of hemecontaining globin polypeptides (A2B2) in red cells. Both bind oxygen. Myoglobin oxygen binding curve is a steep hyperbolic curve that indicates a high oxygen binding affinity even at 10-20 mm-Hg pO2 that ensures mitochondria are supplied evenly and well. Oxygenation and deoxygenation of Hb takes place in stages: Hb 4 <=> Hb4O2 <=>Hb4O4 <=> Hb4O6 <=> Hb4O8 Hemoglobin’s sigmoid curve indicates a positive cooperative binding among the four subunits that increases toward saturation such that each oxygen bound increases the binding affinity of the unoccupied hemes for oxygen. [The Hill coefficient (exp n) of the Hill equation (y/100 = Kxn/(1 + Kxn) is 2.5 (at pCO2 of 40mm, plasma pH 7.44) indicating each Hb tetramer averages about 2.5 molecules of bound oxygen and changes in pO2 account for sufficient oxygen exchange to accommodate respiratory needs. For myoglobin n = 1, for the hyperbolic curve, no cooperative binding consistent with mass action.] 199 Porphyrins contain four pyrroles, each with differing side chains (see Baynes, Fig 27.4). Two 5-ALA (from the mitochon-drion) condense in the cytosol to yield PBG (porphobilinogen), then 4 PBG condense to form uroporphyrinogen III, to co-porphyrinogen III, and back into the mitochondrion as protoporphyrinogen IX, then to protoporphyrin IX and finally to heme. 200 Addition of ferrous iron via ferrochelatase to protoporphyrin IX yields metalloporphyrin called heme, in the final step of heme synthesis within the mitochondria. 201 The steep hyperbolic oxygen binding curve of myoglobin indicates high affinity even at low pO2 (10mm, 80% sat.) needed to store a maximum amount of oxygen inside muscle cells for release to mitochondria. Hemoglobin’s sigmoidal binding curve is ideal for facile oxygen exchange required for transport (binding, dissociation) of oxygen between lungs and peripheral tissues. 202

The g-carboxylate group provides a spatial geometry for the negative charge of both groups to bind Ca2+ cation in a coordinate complex. 203 The function of N-terminal cluster of chelated calcium ions (about 10-12) of the coagulation zymogen helps it to bind to exposed negative charges of phospholipids membranes ruptured in endothelial cells that line blood vessels. 204 The extremely dilute factors in circulation are concentrated together in close proximity by their electrostatic binding to the exposed phospholipids. Concentration and proximity of substrate zymogen and activating protease greatly facilitates the individual rates and therefore the zymogen cascade series activation rates even more. 205 Osteoclastin has similar vitamin K-dependent γ-carboxyglutamate calcium chelating clusters suggesting a role for vitamin K deficiency in osteoporosis. 206 Oxalate (HOOC-COOH). Alpha: malonate (HOOC-CH2-COOH); Beta: succinate, citrate. Gamma: a-KG. Delta: ? 207

Glycolysis; bisphosphoglycerate mutase converts 1,3-BPG to 2,3-BPG in near molar equivalence to hemoglobin subunits. The 2,3BPG facilitates the oxygenation of Hb in the lungs and dissociation of oxygen in the tissue capillaries. 2,3-BPG is degraded to 3-PG via bisphosphoglycerate phosphatase, which is the next substrate in glycolysis after 1,3BPG. 208 DeoxyHHb has a lower affinity than Hb-O2 thereby assisting oxygen gas diffusion into tissue cells after oxygen dissociation. 209 Carbon dioxide reacts with water via carbonic anhydrase to yield carbonic acid, which dissociates into H+ and bicarbonate. The resulting increase in acidic protons is taken up by Hb-O2 , which has a higher pKa yielding HHb-O2, which has lower affinity for oxygen thereby yielding deoxyHHb, which circulates back to the lungs. The other molecule is 2,3-BPG, a negative allosteric effector of oxygen affinity for Hb. Putting the two negative allosteric effectors together, H+ and 2,3-BPG, facilitates both oxygen dissociation from hemoglobin in the capillaries and diffusion of oxygen into cells. 210 The carbon dioxide reacts with each N-terminal of the β-chains to form a carbamino –NH-COO– group. Carbamino-Hb accounts for about 13-15% and carbamino-plasma proteins is about 4% or about 17-19% of the CO2 carriage is carbamino-protein. Dissolved CO2 is about 3.5%; HCO3– is about 80% (RBC + plasma). All together CO2 transport from peripheral tissues is accounted for.

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NBDE Part I Biochem Review and Study Guide

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