BIOCHEMISTRY - Laboratory Practical - NAU 2015

MODULE II

ENZYMES AND METABOLIC PATHWAYS.

Laboratory Work 8

EXPERIMENTAL VERIFICATION OF THE CITRIC ACID CYCLE FUNCTIONING

Objective: to master the method for experimentally confirming the functioning of The Citric Acid Cycle via The production of CO₂.

Basic Theoretical Background

The Citric Acid cycle (CAC) is the primary metabolic pathway of METABOLISM/26.html">Energy Metabolism. The substrate for the CAC is acetyl-CoA, which is formed mainly through the β-oxidation of Higher Fatty acids or The oxidative decarboxylation of Pyruvate.

The Reactions of the CAC are cyclic, and intermediates can be utilized not only in Catabolic pathways but also in anabolic processes (e.g., for the synthesis of Amino Acids and glucose). Therefore, the CAC is an amphibolic process.

The CAC includes four dehydrogenase reactions, in which Coenzymes are reduced to serve subsequently as substrates for the Respiratory Chain, as well as two decarboxylation reactions that release one molecule of CO₂ each. Gas evolution is one of the proofs of CAC functioning.

Equipment: test tubes, pipettes, porcelain mortar and pestle, Glass sand, cotton wool, funnel, pH meter, thermostat, balance, gas burner.

8.1. Verification of mitochondrial citric acid cycle functioning via CO₂ production

Materials and Reagents: enzyme preparation containing CAC and pyruvate oxidative decarboxylation Enzymes (5 g of baker's Yeast is ground in a porcelain mortar with 2–3 ml of distilled Water and a small amount of glass sand, 8–10 ml of water is added, and the mixture is filtered through cotton wool), 1/15 M phosphate buffer, pH = 7.4; pyruvic acid or 5% sodium pyruvate, Ca(OH)₂ suspension, 0.1 M H₂SО₄ solution.

Procedure

The conversion of acetyl-S-CoA in the presence of Cell/35.html">Mitochondria containing CAC enzymes is accompanied by The formation of CO₂. Pyruvic acid is used as the source of acetyl-S-CoA entering the CAC; under the action of the mitochondrial multienzyme pyruvate dehydrogenase complex, it undergoes oxidative decarboxylation to yield acetyl-S-CoA.

Two test tubes — control and experimental — are filled with reagents according to Table 8.1.

To bind the produced CO₂, Ca(OH)₂ is added to the incubation medium. Upon completion of incubation, the bound CO₂ is detected by the evolution of gas bubbles formed after The addition of sulfuric acid to the incubation medium.

Class="right">Table 8.1

Contents of the control and experimental test tubes

Processing of Experimental Data

Write out all CAC reactions, labeling the CAC intermediates, enzymes, substrates, and reaction products. Construct a table indicating the enzyme class to which each CAC enzyme belongs. Derive the overall equation for the CAC. List the decarboxylation and dehydrogenase reactions, and name the pyridine-linked and flavin-linked dehydrogenases. Write the structures of the coenzymes involved in the dehydrogenase reactions in both their oxidized and reduced forms. Construct a table indicating the source of each of the eight hydrogen atoms attached to the coenzymes upon their reduction in the CAC reactions.

Selection/5.html">Control Questions and Tasks

1. What is the Intracellular Localization of CAC enzymes?

2. Explain the Structure AND Functions of mitochondria.

3. Why is it necessary to bind CO₂ during the procedure and then release it via reaction with sulfuric acid? Write the equations for the corresponding reactions.

4. What Competitive Inhibitors of succinate dehydrogenase do you know? How can The Effect of a competitive inhibitor be reversed?

5. Define the rate-limiting reaction of a metabolic pathway. Name the rate-limiting reaction of the TCA cycle.

6. Prove that the TCA cycle is an amphibolic pathway.

7. What are anaplerotic reactions? Which reactions serve as anaplerotic reactions for the TCA cycle?

8. What Vitamins are essential for the functioning of the TCA cycle?

9. Explain the functioning of the $\alpha$-ketoglutarate dehydrogenase complex. What Enzymes and Coenzymes does it contain?

References: [1; 4—7].