BABS 2244 Metabolic Biochemistry Group Report Date: 10 July 2014 Name Chay Min Jie Choong Mel June Khoo Kar Hoe
Student ID 14WAR10522 14WAR10521 14WAR08406
Title: Acyl Activation Reactions Objective: 1. To determine the actual moles of hydroxamate formed. 2. To determine the compound activated in each assay tube per hour. 3. To determine the optical density for acyl activations. 4. To test the expected purity of crude homogenate enzyme. Introduction: Enzyme released by respiration is in part conserved as chemical energy in the form of ATP. Among other functions this ATP, is used by to drive many endergonic reactions. For examples, synthesis of acetyl-CoA and synthesis of peptide bond in proteins or peptides. Proper synthesis and breakdown of molecules cont aining carboxylic acids is a vital part of metabolism in all living organisms. Given the relatively inert chemical nature of many carboxylic acids, activation is a necessary step prior to use in the various anabolic and catabolic pathways that utilize these acids. Lipids, amino acids, sugars, cutin, suberin, glucosinolates, and various other secondary metabolites are built in part using activated carboxylic acids. There is immense variation in the size and structure of organic acids; it is not surprising that most organisms have evolved large families of enzymes that activate them. Collectively, these enzymes use a variety of compounds to activate the carboxylate group; however, the largest of the enzyme families is the acid-thiol ligases. The most common thiol compound used in these reactions is coenzyme A, and the enzymes are generally categorized as CoA ligases or CoA synthetases. Acetate Activation (aceto-CoA kinase): Several different pathways are now known for the activation of acetate. One of these, found thus far only in certain microorganisms is initiated by the phosphoryIation of acetate with ATP by aceto-kinase, followed by the th e transfer of the acetyl group to CoA by the action of phosphotransacetylase ATP + acetate → acetyl phosphate + ADP Acetyl phosphate + CoA → acetyl CoA + phosphate In animal tissues, yeast, plants and Rhodospirihm rubrum, another pathway of acetate activation. This involves a reaction of ATP, acetate, and CoA, resulting in a split of ATP with the formation of acetyl CoA, A5P, and PP, and has been termed the aceto-CoA-kinase reaction.
ATP + acetate + CoA ↔ acetyl CoA + A5P + PP The CoA synthetase reaction proceeds through a two-step mechanism involving the conversion of the carboxylate and ATP to an enzyme-bound carboxyl-AMP intermediate (called an adenylate) with the release of pyrophosphate (PPi). Then, the activated carbonyl carbon of the adenylate is coupled to the thiol of CoA, followed by enzyme release of the thioester and AMP. Amines or alcohol groups can also act as the nucleophile in this reaction. While both steps of the reaction are reversible, and have been demonstrated in vitro, PPi hydrolysis by pyrophosphatase in vivo drives the equilibrium to the right, and makes the reaction irreversible: Enz + acetate + ATP ↔ Enz – AMP – acetate + PPi Enz - AMP + acetate + CoA ↔ acetyl CoA + AMP + Enz Enz Acetate +ATP + CoA ↔ acetyl-CoA +AMP + PPi Amino acid (AA) activation: The initial step in the synthesis of proteins is assumed to be an activation of the carboxyl group of the free amino acid similar to the mechanism of acetate activation. The reaction results in the formation of an enzyme-AMP-amino acid complex in which the 5’ phosphate of AMP is linked to the carboxyl of the amino acid as a mixed anhydride. Enz + ATP + AA ↔ Enz – AMP – AA + PPi The activated amino acid at the state of an acyl 5’ adenylate is now capable of non endergonic transfers to many possible receptors. If these are ac yl transfers, the activation energy initially donated by the hydrolysis of ATP will be conserved. The transferred amino acid wil still be capable of forming a peptide bond at any transfer level. The initial acceptor of the activated amino acid may be some form of soluble RNA. This has been determined by the finding of a non particulate RNAase sensitive acceptor of the activated amino acid. This can be measured by an amino acid dependent exchange of AMP into ATP. Soluble RNA plays a role in protein synthesis. The preparation of rat liver that contains a labeled amino acid bonded to a soluble RNA can transfer the labeled amino acid to a microsomal preparation in the presence of guanosine triphospahte (GTP). These steps were summarized as follow:
Explanation: First step is amino acid activation reaction. An activating enzyme (aminoacyl-RNA synthetase) specific for one f the amino acids found in proteins catalyzes a reaction between carboxyl group of the amino acid and pyrophosphoryl group of ATP. With the elimination of inorganic pyrophosphate, a mixed anhydride is formed between carboxyl group of amino acid and 5’ phosphate of AMP. This amino acyl adenylate remains firmly attached to the enzyme surface and does not dissociate or accumulate as a free intermediate. Methods: 1. The following mixtures were prepared in different test tubes. Substance Tube Number 1 2 3 4 5 6 7 8 0.02M Mix of 15L-amino acid 0.2 0.2 0.2 1.0 KF Potassium acetate 0.2 0.2 0.2 0.1M ATP 0.3 0.3 0.3 0.1M Tris-Cl 0.6 0.6 0.6 0.6 3.0M NH2OH.HCl 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.1M MgCl2 0.3 0.3 0.3 0.3 0.01M Hydroxamate standard 0.1 0.2 0.4 H2O 2.0 1.9 1.8 1.6 0.2 0.2 0.3
9 0.3 0.6 1.0 0.3 0.4
10 0.6 1.0 0.3 0.7
Standard tubes (Tubes 1 to 4) 2. For standard tubes (tubes 1 to 4), 1.4ml of cool 10% TCA was added in. 3. Then, the contents of tubes 1 to 4 were mixed thoroughly. 4. After that, 0.6ml of 2M FeCl3 was added into each tube. 5. The tubes were allowed to sit for 10 minutes. 6. The optical densities of tubes 2 to 4 were read and recorded at 520nm against the blank (tube 1). Enzyme assay tubes (Tubes 5 to 10) 2. 0.4ml of liver homogenate was added to enzyme assay tubes (tubes 5 to 10). 3. Then, the contents of tubes 5 to 10 were mixed thoroughly and incubated for 60 minutes at 37˚C in a water bath. 4. After 60 minutes, 1.4ml of cool 10% TCA was added into each tube to stop the reaction. 5. Then, the contents of tubes 5 to 10 were mixed thoroughly. 6. After that, 0.6ml of 2M FeCl3 was added into each tube. 7. The coagulated protein in tubes 5 to 10 was centrifuged and the clear supernatants were decanted. 8. The clear supernatants of tubes 5 to 10 were left to sit for 5 minutes. 9. Then, the optical densities of tubes 5 to 10 were read and recorded at 520nm against the blank (tube 1).
Results: Standard Tubes Tubes Volume of 0.01M Hydroxamate Standard 1 2 0.1 3 0.2 4 0.4
Optical Density (520nm) (as blank) 0.127 0.286 0.490
Standard Curve Graph of Optical Density (520nm) against Volume of Hydroxamate 0.5 0.49 0.48 0.47 0.46 0.45 0.44 0.43 0.42 0.41 0.4 0.39 0.38 0.37 0.36 0.35 0.34 0.33 ) 0.32 m0.31 n 0.3 00.29 2 5 ( 0.28 y0.27 t 0.26 i s 0.25 n0.24 e D0.23 l 0.22 a0.21 c i t 0.2 p0.19 O0.18 0.17 0.16 0.15 0.14 0.13 0.12 0.11 0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0
y = 1.1836x + 0.0245 R² = 0.9867
0
0.05
0.1
0.15
0.2
0.25
0.3
Volume of Hydroxamate
0.35
0.4
0.45
Enzyme Assay Tubes (from the graph) Tubes Volume of 0.01M Hydroxamate Standard 5 0.065 6 0.053 7 0.075 8 0.043 9 0.048 10 0.032
Optical Density (520nm) 0.100 0.187 0.114 0.075 0.080 0.062
Discussion: Each of the test tubes 5, 7 and 8 has 0.02 M Mix of 15L amino acid. In the amino acid activation, the aminoacyl-tRNA synthetase (enzyme) attracted an amino acid, ATP. The active site bound the Amino Acid and ATP. The ATP then lost two phosphate groups and then joined the amino acid in the form of AMP. It formed enzyme AMP – AA complex. The tRNA then covalently bonds to the amino acid, taking the place of the AMP, thus an AMP is lost. The high volume of hydroxamic acid was found in tubes 5, 7 and 8 compared to test tube 9 and 10. The volume of hydroxamic acid in test tube 6 is lower than test tube 5. Each of the test tubes 6, 7 and 8 has 1.0M KF Potassium acetate. In the acetate activation, The AMP-forming acetyl-CoA synthetase complex is formed. First, AMP must be bound by the enzyme to cause a conformational change in the active site, which allows the reaction to take place. The active site is referred to as the A-cluster. A crucial lysine residue must be present in the active site to catalyze the first reaction where Co-A is bound. Co-A then rotates in the active site into the position where acetate can covalently bind to CoA. The covalent bond is formed between the sulfur atom in Co-A and the central carbon atom of acetate. The high volume of hydroxamic acid of tubes 6, 7, 8 than compared to test tube 9 and 10. In the test tube 5, 6, 7 and 9 , the ATP is the high-energy molecule and helps to form complex in acyl activation. Therefore, the tubes have high absorbance value in hydroxamic acid which can be found in the product of the reaction. The tube 7 has the highest volume. The presence of ATP acts as activator of forming a complex Enz-AMP-acetate or Enz-AMP-AA. In tube 9 has no amino acid or potassium acetate added in the reaction but it formed hydroxamic acid which can be found in the liver enzyme. The liver enzyme consisted of amino acid or potassium acetate which can promote the acyl activation. The test tube 8 and 10 has no ATP added; therefore it has low volume of hydroxamic compared to the rest of test tubes. The ATP can promote the reaction. Based on the results obtained from the experiment, we can make comparison between Tube 5, 6 and 9. Tube 5 only has the presence of 15L-amino acid, while Tube 6 only have potassium acetate and Tube 9 only have liver homogenate in it. Tube 5 produced 0.065ml of hydroxamate, while Tube 6 produced 0.053ml of hydroxamate and Tube 9 only produced 0.048ml of hydroxamate. Tube 9 which consists of rat liver homogenate has the lowest volume of hydroxamate produced because it is a crude and unrefined sample. Therefore, although acetate and amino acid can be found in it, their relative volume is quite low. Both Tube 5 and 6 have a higher number of hydroxamate because they are from a refined source that is produced specifically with a fixed number of moles and concentration.
Besides that, we can also compare between Tube 9 and 10. Both Tube 9 and 10 only have liver homogenate, which no other substrate in it, but ATP is supplied in Tube 9 while Tube 10 is deprived of it. Therefore, we can see that Tube 9 have a higher volume of 0.01M hydroxamate produced, which is 0.048ml compared to Tube 10 which only produced 0.032ml of hydroxamate due to the absence of ATP. However, Aminoacyl-tRNA synthetase enzymes utilize ATP as an energy source to attach a tRNA molecule to its specific amino acid, forming an aminoac yl-tRNA complex, ready for translation at ribosomes. The energ y is made available by ATP hydrolysis to adenosine monophosphate (AMP) as two phosphate groups are removed. Therefore, we can say that amino acid is essential in order for the reaction to occur and intermediate to form. Even though ATP is not supplied to Tube 10, we can still see the production of hydroxamate because ATP is present in small amount in the fresh liver homogenate, where it can be located in the liver’s mitochondria. When comparing Tube 7 and Tube 8, we can see that there is a significant difference in the volume of hydroxamate produced between the two tubes. Tube 7 produced 0.075ml of hydroxamate while Tube 8 produced 0.043ml of hydroxamate. Both tubes consisted of all the three substrates (amino acid, potassium acetate and liver homogen ate), while Tube 7 is supplied with ATP but not in Tube 8. ATP acts as an activator for both acetate activation and amino acid activation; therefore, the presence of ATP helps to promote the formation of Enz-AMP-acetate intermediate state and leads to a higher activation to produce a higher volume of hydroxamate. However, reaction still occurs in Tube 8 even though ATP is not supplied because small amount of ATP is present in the mitochondria of the liver homogenate. Therefore, due to the less amount of ATP present in Tube 8, the activation reaction is slower compared to Tu be 7 and less hydroxamate is produced. Conclusion: Based on the result obtained, Tube 7 that have all three substrates with ATP provided have the highest activation which leads to the highest volume of hydroxamate produced. References: 1. Pratt C.W., Cornely, K. Essential Biochemistry. John Wiley & Sons, Inc. (2004) 2. Brown TD, Jones-Mortimer MC, Kornberg HL (1977) The enz ymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol 102:327 – 336
