Module No.
BIOCHEMISTRY I
Urea Cycle, Amino Acid Degradation and Synthesis Dr. Pacifico E. Calderon, MD
8.1.2 Feb.15, 17
OUTLINE I. UREA CYCLE II. BIOSYNTHESIS OF UREA III. AMINO III. AMINO ACID DEGRADATION DEGRADATION AND AND SYNTHESIS SYNTHESIS IV. CLINICAL CORRELATES LEGEND: Black = PPT, Red = PPT side notes, notes, blue = book
Read, Understand and memorize all including the table!
UREA CYCLE
Urea is the major disposal form of amino groups o Accounts for 90% of N-containing compounds in the urine) Pathway for removal of nitrogenous waste products in the body Present only in the liver Urea is formed in the liver then enters the blood and excreted in urine. Remember that urea is the major disposal form of amino groups derived from amino acids. 1 nitrogen of urea is from NH3 (ammonia) and other is from aspartate Glutamate is the immediate precursor of ammonia and aspartate nitrogen Organs where transamination can take place o Liver o Kidneys o Muscles st 1 2 reactions (leading to synthesis of urea) occur in the mitochondria and mitochondria and the rest are in the cytosol
CARBAMOYL PHOSPHATE SYNTHASE I
DONORS OF THE ATOM OF UREA
IN SUMMARY NH3 From free ammonia NH3 from aspartate CO2
BIOSYNTHESIS OF UREA
Transers: Adriano, Atienza, Choy
Checked by: Gayados
Urea is the major end product of of nitrogen catabolism in humans Synthesis of 1 mol of urea requires o 3 mol of ATP of aspartate, and o 1 mol each ammonium ion and employs five enzymes Of the six participating amino acids function solely as an enzyme o N-acetylglutamate activator The others others serve as carriers of the atoms that ultimately become urea Urea synthesis is a cyclic process While ammonium ion, CO2, ATP and aspartate are consumed ornithine consumed in reaction of ornithine o The carbamoyl transferase (ornithine transcarbamoylase) transcarbamoylas e) and regenerated in arginase (argininosuccinase arginase) o There thus is no net loss or gain of ornithine, citrulline, argininosuccinate, or arginine o Some reactions of urea synthesis occur in the matrix of the mitrochondria, and other reactions in the cytosol
Carbamoyl Phosphate Synthase Synthase I initiates urea biosynthesis o RATE LIMITING ENZYME OF THE UREA CYCLE o Active only in the presence of N-acetylglutamate An allosteric activator that enhances the affinity of the synthase for ATP Condensation of CO2, ammonia and ATP to form carbamoyl phosphate is catalyzed by mitochondrial o Carbamoyl phosphate synthase I A cytosolic cytosolic form form of this enzyme o Carbamoyl phosphate synthase II Uses glutamine rather ammonia ammonia as the nitrogen donor and functions in pyrimidine biosynthesis The concerted action of glutamate dehydrogenase and carbamoyl phosphate synthase I o Shuttles amino nitrogen into cabamoyl phosphate Compound with high group transfer potential Synthesis of 1 mol of carbamoyl phosphate o Requires 2 mol of ATP 1st ATP – – phosphoryl donor for formation of the mixed acid anhydride bond of carbamoyl phosphate nd 2 ATP – – provides provides driving force for synthesis of the amide bond of carbamoyl phosphate o The other products are 2 mol of ADP and 1 mol of P 1 Reaction of HCO3 with ATP forms o Carbonyl phosphate o ADP Ammonia then displaces ADP forming o Carbamate and orthophosphate nd Phosphorylation n of carbamate by the 2 ATP then o Phosphorylatio forms CARBAMOYL PHOSPHATE
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L-ORNITHINE TRANSCARBAMOYLASE
Carbamoyl phosphate + Ornithine = Citrulline L-Ornithine transcarbamoylase catalyzes transfer of the carbamoyl group of carbamoyl phosphate to ornithine forming o Citrulline and Orthophosphate While the reaction occurs in the mitochondrial matrix, both the formation of ornithine and the subsequent metabolism of citrulline take place in the o CYTOSOL Entry of ornithine into mitochondria and exodus of citrulline from mitochondria therefore o Involve mitochondrial inner membrane permeases
o
ARGININOSUCCINATE SYNTHASE
Cituilline + Aspartate = Argininosuccinate Argininosuccinate synthase links aspartate and citrulline via the amino group of aspartate and provides the nd nitrogen of urea o 2 The reaction requires ATP and involves intermediate formation of citrullyl-AMP o Subsequent displacement of AMP by aspartate then forms argininosuccinate.
ARGININOSUCCINATE LYASE
Cleavage of argininosuccinate forms arginine and fumarate catalyzed by argininosuccinate lyase The reaction proceeds with retention of all 3 Nitrogens in arginine and release of the aspartate skeleton as fumarate Subsequent addition of water to fumarate forms o L-malate + Whose subsequent NAD -dependent oxidation forms oxaloacetate These two reaction are analogous to reactions of the citric acid cycle o But are catalyzed by cytosolic Fumarase Malate dehydrogenase of oxaloacetate by glutamate o Transamination aminotransferase then re-forms aspartate The carbon skeleton of aspartate-fumarate thus acts as a carrier of the nitrogen of glutamate into a precursor of urea
MNEMONIC ORDINARY CARELESS CRAPPERS ARE ALSO FRIVOLOUS ABOUT URINATION o Ornithine + carbamoyl phosphate = Citrulline o Citrulline + Aspartate = Argininosuccinate o Argininosuccinate = Fumarate + Arginine Fumarate (converted to malate TCA) o Arginine = Urea + ornithine Immediate precursor of urea
ARGINASE
FATE OF UREA Diffuses from the liver and is transported in the blood to the kidneys, where it is filtered and excreted in the urine A portion of urea diffuses from the blood into the intestine, and is cleaved to CO2 and NH3 by bacterial urease CLINICAL CORRELATES
Cleave of arginine release urea and re-forms ornithine Cleavage of the guanidine group of arginine, catalyzed by o LIVER arginase Releases urea The other product, ornithine, reenters liver mitochondria and participates in additional rounds of urea synthesis Ornithine and lysine are potent inhibitors of arinase, and compete with arginine. Arginine also serves as the precursor of the potent muscle relaxant 2+ Oxide (NO) in a Ca -dependent reaction o Nitric catalyzed by NO synthase
N-ACETYLGLUTAMATE SYNTHASE ACETYLGLUTAMATE DEACYLASE
NH3 Aspartate o CO2 Rate Limiting Step o Reaction: CO2 + NH3 Carbamoyl phosphate o Enzyme: Carbomyl Phosphate Synthetase I (CPS-1) CPT-1 = Beta-oxidation of fats CPS-1 = Urea cycle CPS-2 = Biosyntehsis of pyrimidines Energy Requirement o 4 moles of ATP Co-Factors o N-acetylglutamate – the allosteric activator of CPS-1 o Biotin – for carboxylation reaction Arginosuccinate synthethase: 3rd final ATP consumption Arginase: cleaves arginine to ornithine and urea; occurs almost exclusively in the liver; hence urea cycle does not occur anywhere else Where will we encounter carbamoyl phosphate? In biosynthesis of pyrimidines Mitochondria Carbamoyl Phosphate (via action of CPS I) can’t diffuse to the cytoplasm, it must first be converted to citrulline. Remaining steps in the cytoplasm o
AND
N-
The activity of carbamoyl phosphate synthase I is determine by N-acetylglutamate o Whose steady-state level is dictated by t he balance between its Rate of synthesis from acetyl-CoA and glutamate Rate of hydrolysis to acetate and glutamate N-acetylglutamate synthase + l-glutamate → N-acetyl-l-glutamate + o Acetyl-CoA CoASH N-acetylglutamate deacyclase (hydrolase) o N-acetyl-l-glutamate + H2O → l-glutamate + acetate
HEREDITARY HYPERAMMONEMIA Enzyme defect in the urea cycle o Type 1: Carbamoyl phosphate synthetase I o Type 2: Ornithine transcarbamoylase (most common urea cycle deficiency; neurologic abnormalities) Causes o Hyperammonemia o Elevated blood glutamine o Decreased BUN (Blood urea nitrogen) Present with vomiting, hyperventilation, convulsions, o Lethargy, cerebral edema, coma, death Treat with o Low protein diet o Administration of sodium benzoate or phenylpyruvate to capture and excrete excess nitrogen
ACQUIRED HYPERAMMONEMIA Compromised liver function Presents with tremors, slurring speech, somnolence (sleepiness), vomiting, cerebral edema and blurring of vision
AMINO ACID DEGRADATION AND SYNTHESIS CATABOLISM
UREA CYCLE SUMMARY Substrates/ Raw Materials
BOTH KETOGENIC AND GLUCOGENIC FYI double You = FYIW
Transers: Jandel, Kyle, Pat
Checked by: Billy
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Biochemistry I
Urea Cycle, Amino acid degradation and synthesis
F = Phenylalanine Y = Tyrosine o I = Isoleucine o W = Tryptophan o o
KETOGENIC Produce ketone bodies, it cannot give rise to glucose, not a substrates for gluconeogenesis and cannot give rise to the net formation of glucose
Amino acid whose catabolism yield o Acetoacetate o Acetyl-CoA / acetoacetyl-CoA Amino acids o Leucine o Lysine What are the AA that does not undergo transamination for the removal of the alpha amino group? o Lysine and threonine
Module #8, Lecture #1.2
Alanine Aspartate Glutamate
Transamination of a-ketoacids
Glutamine Asparagine
Amidation of glutamate and aspartate
Proline
Synthesized from glutamate
Cysteine
Made from methionine and serine
Serine
Made from 3-phosphoglycerate
Glycine Tyrosine
Made from serine Made form phenylalanine
SPECIALIZED PRODUCTS OF AA Amino Acids
GLUCOGENIC Produce glucose Substrate for gluconeogenesis gives net rise to glucose or glucogen Amino acid whose catabolism yield o Pyruvate or Intermediates of the Kreb Cycle Glucose via gluconeogenesis Glycogen in muscle or liver Amino acids o “All others”
Raw Material in Biosynthesis
Glycine
Heme Purines Creatine Also conjugated to Bile acids
Serine
Phospholipid and sphingolipid Purines Thymine
Glutamate
GABA
Cysteine
Thioethanolamine of CoA Taurine
Histidine
Histamine
Arginine
Creatinine Polyamines Nitric Oxide
Tryptophan
Serotonin Niacin Melatonin
Tyrosine
Cathecolamines Thyroid hormones (T3 and T4) Melanin
SUMMARY TABLE Ketogenic Leucine Lysine
Ketogenic and Glucogenic Phenylalanine Tyrosine Tryptophan Isoleucine
Glucogenic
All Others
GLUCOGENIC AA
CLINICAL CORRELATES
Entry Point a-ketoglutarate “na GG PAH!”
Amino acids Glutamine Glutamate Proline Arginine Histidine
Pyruvate “AlaS Grade Conscious TT”
Alanine Serine Glycine Cysteine Threonine Tryptophan
Fumarate “Phewww T y!” Succinyl CoA “Meet Valentines, Tulog Oxaloacetate “Asp Asp!”
UREA CYCLE DISORDERS
Phenylalanine Tyrosine Iyak,
Methionine Valine Isoleucine Threonine
Aspartate Asparagine
SYNTHESIS OF NON-ESSENTIAL AA Amino Acids
Synthesized from
Transers: Adriano, Atienza, Choy
There are five well documented diseases of the urea cycle. They each represent a defect in the biosynthesis of enzymes in the urea cycle. These disorders are characterized by: o Hyperammonemia o Encephalopathy o Respiratory alkalosis Four of the five metabolic diseases result in the accumulation of precursors of urea, principally ammonia and glutamine. Ammonia intoxication is most severe when the metabolic block occurs at reactions 1 or 2 (Figure 28 –16), for if citrulline can be synthesized, some ammonia has already been removed by being covalently linked to an organic metabolite. Clinical symptoms common to all urea cycle disorders include o Vomiting o Avoidance of high-protein foods o Intermittent ataxia o Irritability o Lethargy o Severe mental retardation The most dramatic clinical presentation occurs in full-term infants who initially appear normal, then exhibit progressive lethargy, hypothermia, and apnea due to high plasma ammonia levels. The clinical features and treatment of all five disorders are similar
Checked by: Gayados
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Significant improvement and minimization of brain damage can accompany a low-protein diet ingested as frequent small meals to avoid sudden increases in blood ammonia levels. o The goal of dietary therapy is to provide sufficient protein, arginine, and energy to promote growth and development while simultaneously minimizing the metabolic perturbations o
Unlike other urea cycle disorders, the first symptoms of hyperargininemia typically do not appear until age 2 to 4 years. o Blood and cerebrospinal fluid levels of arginine are elevated. The urinary amino acid pattern, which resembles that of lysine-cystinuria, may reflect competition by arginine with lysine and cysteine for reabsorption in the renal tubule.
CARBAMOYL PHOSPHATE SYNTHASE I
AMINO ACID DISORDERS
N-acetylglutamate is essential for the activity of carbamoyl phosphate synthase I Defects in this enzyme results to a rare disease termed Hyperammonemia Type I
N-ACETYLGLUTAMATE SYNTHASE I Catalyzes the formation of N-acetylglutamate from acetylCoA and glutamate Clinical and biochemical features of NAGS deficiency are indistinguishable from the those of carbamoyl phosphate synthase I but, deficiencies from NAGS may respond to administered N-acetylglutamate
ORNITHINE PERMEASE Mutations of the ORNT1 gene that codes for the mitochondrial membrane ornithine permease leads to hyperornithinemia, hyperammonemia, homocitrullinuria syndrome (HHH syndrome) The failure to import cytosolic ornithine into the mitochondrial matrix renders the urea cycle inoperable, with consequent hyperammonemia, and hyperornithinemia due to the accompanying accumulation of cytosolic ornithine. In the absence of its normal acceptor (ornithine), mitochondrial carbamoyl phosphate carbamoylates lysine to homocitrulline, resulting in homocitrullinuria.
PHENYLKETONURIA
ORNITHINE TRANSCARBAMOYLASE
The X-chromosome linked deficiency termed “Hyperammonemia type 2” reflects a defect in ornithine transcarbamoylase. The mothers also exhibit Hyperammonemia and an aversion to high-protein foods. Levels of glutamine are elevated in blood, cerebrospinal fluid, and urine, probably as a result of enhanced glutamine synthesis in response to elevated levels of tissue ammonia.
ARGININOSUCCINATE SYNTHASE
In addition to patients who lack detectable argininosuccinate Synthase activity, a 25-fold elevated Km for citrulline has been reported. In the resulting citrullinemia, plasma and cerebrospinal fluid citrulline levels are elevated, and 1 to 2 g of citrulline are excreted daily.
ARGININOSUCCINATE LYASE
Argininosuccinic aciduria, accompanied by elevated levels of Argininosuccinate in o Blood o Cerebrospinal fluid o Urine Associated with friable, tufted hair (trichorrhexis nodosa) Both early- and late-onset types are known. The metabolic defect is in argininosuccinate lyase. Diagnosis by the measurement of erythrocyte argininosuccinate lyase activity can be performed on umbilical cord blood or amniotic fluid cells.
ARGINASE
Hyperargininemia is an autosomal recessive defect in the gene for arginase.
Transers: Jandel, Kyle, Pat
Checked by: Billy
Most common clinically encountered inborn error of AA metabolism Caused by a deficiency of phenylalanine hydroxylase o Phenylalanine tyrosine In PKU, there is ↓phenylalanine hydroxylase or ↓tetrahydrobiopterin cofactor Tyrosine becomes essential and phenylalanine builds up, leading to excess phenylketones in urine: o Phenylacetate o Phenyllactate o Phenylpyruvate Findings: o Mental retardation o Growth retardation (decreased thyroid hormones) o Fair skin (albinism; decreased melanin) o Eczema o Musty body odor (due to phenylketones) Treatment: ↓phenylalanine and ↑tyrosine in diet Less Tyrosine= Less Tyrosine Derivatives o True - Tyrosine o Love – L-Dopa o Does - Dopamine Page 4 of 6
Biochemistry I
Urea Cycle, Amino acid degradation and synthesis
Not - Norepinephrine Exist - Epinephrine o To – Thyroid hormones o Me – Melanin (not melatonin) o
o
ALKAPTONURIA
Congenital deficiency of homogentisic acid oxidase in the degradative pathway of tyrosine Resulting alkapton bodies cause urine to turn black on standing o Oxidation of homogentisic acid Also, the connective tissue is dark (ochronosis) Benign disease but may have debilitating arthralgias
MAPLE SYRUP URINE DISEASE
Blocked degradation of branched amino acids (isoleucine, valine, leucine) due to a deficiency in branched chain αketoacid dehydrogenase Causes an ↑α-ketoacid in the blood, especially leucine Causes severe CNS defects, mental retardation and death
Homocystinuria
The first step in AA degradation is the removal of N In terrestrial vertebrates, ammonium is converted into urea C atoms of degraded AAs emerge as major metabolic intermediates Inborn errors of metabolism can disrupt AA degradation Human subjects degrade 1% to 2% of their body protein daily at rates that vary widely between proteins and with physiologic state. Key regulatory enzymes often have short half-lives. Proteins are degraded by both ATP-dependent and ATPindependent pathways. Ubiquitin targets many intracellular proteins for degradation. Liver cell surface receptors bind and internalize circulating asialoglycoproteins destined for lysosomal degradation. Polyubiquitinated proteins are degraded by proteases on the inner surface of a cylindrical macromolecule, the proteasome. Entry into the proteasome is gated by a donutshaped protein pore that rejects entry to all but polyubiquitinated proteins. Fish excrete highly toxic NH3 directly. Birds convert NH3 to uric acid. Higher vertebrates convert NH3 to urea. Transamination channels amino acid nitrogen into glutamate. GDH occupies a central position in nitrogen metabolism. Glutamine synthase converts NH3 to nontoxic glutamine. Glutaminase releases NH3 for use in urea synthesis. NH3, CO2, and the amide nitrogen of aspartate provide the atoms of urea. Hepatic urea synthesis takes place in part in the mitochondrial matrix and in part in the cytosol. Changes in enzyme levels and allosteric regulation of carbamoyl phosphate synthase I by N-acetylglutamate regulate urea biosynthesis. Metabolic diseases are associated with defects in each enzyme of the urea cycle, of the membrane-associated ornithine permease, and of NAGS. The metabolic disorders of urea biosynthesis illustrate six general principles of all metabolic disorders. Tandem mass spectrometry is the technique of choice for screening neonates for inherited metabolic diseases
REVIEW 1.
EXPANDED NEWBORN SCREENING The following disorders are covered by the expanded newborn screening program.
Disorder
Module #8, Lecture #1.2
In which of the following organs can transamination take place? A. Liver only B. Kidneys only C. Muscles only D. A and B only E. All of the above
Deficient Enzyme
ANSWER: E = ALL OF THE ABOVE
Cystathionine Beta Synthase 2.
Hypermethioninemia
Methionine Adenosyltransferase
Maple Syrup Urine Disease
Branched Chain Α-Ketoacid Dehydrogenase
Phenylketonuria
Phenylalanine Hydroxylase
Tyrosinemia Type I
Fumarylacetoacetate Hydrolase
Which of the following reactions can provide free ammonia for the urea cycle? A. Transamination of alanine to pyruvate B. Transamination of aspartate to OAA C. Deamination of glutamine to glutamate D. All of the above ANSWER: C = Deamination of glutamine to glutamate
SOURCES o
Tyrosinemia Type II
Tyrosine Aminotransferase
o o
Dr. calderon’s powerpoint th th Harper 30 Illustrated biochemistry 30 Edition th Urinalysis and body fluids 5 edition by Strasinger
SUMMARY
Proteins are degraded to amino acids, and protein turnover is tightly regulated
Transers: Adriano, Atienza, Choy
Checked by: Gayados
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Biochemistry I
Urea Cycle, Amino acid degradation and synthesis
Module #8, Lecture #1.2
APPENDIX
Transers: Adriano, Atienza, Choy
Checked by: Gayados
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