Citric acid cycle mechanisms

Elucidating the stereochemistry of reaction at prochirality centers is a powerful method for studying detailed mechanisms in biochemical reactions. As just one example, the conversion of citrate to (ds)-aconitate in the citric acid cycle has been shown to occur with loss of a pro-R hydrogen, implying that the reaction takes place by an anti elimination mechanism. That is, the OH and H groups leave from opposite sides of the molecule. 

The first step in the citric acid cycle is reaction of oxaloacetate with acetyl CoA to give citrate. Propose a mechanism, using acid or base catalysis as needed. 

Figure 29.12 MECHANISM The citric acid cycle is an eight-step series of reactions that results in the conversion of an acetyl group into two molecules of C02 plus reduced coenzymes. Individual steps are explained in the text.

Problem 29.11 Write mechanisms for step 2 of the citric acid cycle, the dehj dration of citrate and the addition of water to aconitate. 

Step 2 of Figure 29.13 Decarboxylation and Phosphorylation Decarboxylation of oxaloacetate, a jB-keto acid, occurs by the typical retro-aldol mechanism like that in step 3 in the citric acid cycle (Figure 29.12), and phosphorylation of the resultant pyruvate enolate ion by GTP occurs concurrently to give phosphoenol-pyruvate. The reaction is catalyzed by phosphoenolpyruvate carboxykinase. 

Write a mechanism for the conversion of a--ketoglutarate to succinyl CoA in step 4 of the citric acid cycle (Figure 29.12). 

Ethanol is oxidized by alcohol dehydrogenase (in the presence of nicotinamide adenine dinucleotide [NAD]) or the microsomal ethanol oxidizing system (MEOS) (in the presence of reduced nicotinamide adenine dinucleotide phosphate [NADPH]). Acetaldehyde, the first product in ethanol oxidation, is metabolized to acetic acid by aldehyde dehydrogenase in the presence of NAD. Acetic acid is broken down through the citric acid cycle to carbon dioxide (CO2) and water (H2O). Impairment of the metabolism of acetaldehyde to acetic acid is the major mechanism of action of disulfiram for the treatment of alcoholism. 

NO also has cytotoxic effects when synthesized in large quantities, eg, by activated macrophages. For example, NO inhibits metalloproteins involved in cellular respiration, such as the citric acid cycle enzyme aconitase and the electron transport chain protein cytochrome oxidase. Inhibition of the heme-containing cytochrome P450 enzymes by NO is a major pathogenic mechanism in inflammatory liver disease. 

The reaction involves biotin as a carrier of activated HCO3 (Fig. 14-18). The reaction mechanism is shown in Figure 16-16. Pyruvate carboxylase is the first regulatory enzyme in the gluconeogenic pathway, requiring acetyl-CoA as a positive effector. (Acetyl-CoA is produced by fatty acid oxidation (Chapter 17), and its accumulation signals the availability of fatty acids as fuel.) As we shall see in Chapter 16 (see Fig. 16-15), the pyruvate carboxylase reaction can replenish intermediates in another central metabolic pathway, the citric acid cycle. 

A multiauthor book on the citric acid cycle, including molecular genetics, regulatory mechanisms, variations on the cycle in microorganisms from unusual ecological niches, and evolution of the pathway. Especially relevant are the chapters by H. Gest (Evolutionary Roots of the Citric Acid Cycle in Prokaryotes),... 

Figure 10-6 Reactions of the citric acid cycle (Krebs tricarboxylic acid cycle). Asterisks designate positions of isotopic label from entrance of carboxyl-labeled acetate into the cycle. Note that it is not the two carbon atoms from acetyl-CoA that are immediately removed as C02 but two atoms from oxaloacetate. Only after several turns of the cycle are the carbon atoms of the acetyl-CoA completely converted to C02. Nevertheless, the cycle can properly be regarded as a mechanism of oxidation of acetyl groups to C02. Green daggers (+) designate the position of 2H introduced into malate as 2H from the medium. FADS designates covalently bound 8-histidyl-FAD (see Chapter 15).

One of the first persons to study the oxidation of organic compounds by animal tissues was T. Thunberg, who between 1911 and 1920 discovered about 40 organic compounds that could be oxidized by animal tissues. Salts of succinate, fumarate, malate, and citrate were oxidized the fastest. Well aware of Knoop s (3 oxidation theory, Thunberg proposed a cyclic mechanism for oxidation of acetate. Two molecules of this two-carbon compound were supposed to condense (with reduction) to succinate, which was then oxidized as in the citric acid cycle to oxaloacetate. The latter was decarboxylated to pyruvate, which was oxidatively decarboxylated to acetate to complete the cycle. One of the reactions essential for this cycle could not be verified experimentally. It is left to the reader to recognize which one. 

Since in the citric acid cycle there is no net production of its intermediates, mechanisms must be available for their continual production. In the absence of a supply of oxalacetic acid, acctaic" cannot enter the cycle. Intermediates for the cycle can arise from the carinxylation of pyruvic acid with CO, (e.g., to form malic acid), the addition of CO > to phosphcnnlpyruvic acid to yield oxalacetic acid, the formation of succinic acid from propionic acid plus CO, and the conversion of glutamic acid and aspartic acid to alpha-ketoglutaric acid and oxalacetic acid, respectively. See Fig. 3. 

Systems of biochemical reactions like glycolysis, the citric acid cycle, and larger and smaller sequential and cyclic sets of enzyme-catalyzed reactions present challenges to make calculations and to obtain an overview. The calculations of equilibrium compositions for these systems of reactions are different from equilibrium calculations on chemical reactions because additional constraints, which arise from the enzyme mechanisms, must be taken into account. These additional constraints are taken into account when the stoichiometric number matrix is used in the equilibrium calculation via the program equcalcrx, but they must be explicitly written out when the conservation matrix is used with the program equcalcc. The stoichiometric number matrix for a system of reactions can also be used to calculate net reactions and pathways. 

We have now examined the mechanism of several steps in glycolysis and one in the citric acid cycle and we have seen enough to look at the outline of these two important processes and the link between them (see opposite). 

This is the study of the speed and mechanism of a chemical reaction. A cyclic system and set of chemical pathways within cells to make and use energy. Also known as the citric acid cycle. 

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