Citric acid cycle reactions stereospecificity

Two consecutive reactions of the citric acid cycle (Fig. 10-6), the dehydration of citrate to form czs-aconi-tate and the rehydration in a different way to form isocitrate (Eq. 13-17), are catalyzed by aconitase (aconi-tate hydratase). Both reactions are completely stereospecific. In the first (Eq. 13-17, step a), the pro-R proton from C-4 (stereochemical numbering) of citrate is removed and in step c isocitrate is formed. Proton addition is to the re face in both cases. 

It may be protested that the reaction of the citric acid cycle by which oxaloacetate is converted to oxo-glutarate does not follow exactly the pattern of Fig. 17-18. The carbon dioxide removed in the decarboxylation step does not come from the part of the molecule donated by the acetyl group but from that formed from oxaloacetate. However, the end result is the same. Furthermore, there are two known citrate-forming enzymes with different stereospecificities (Chapter 13), one of which leads to a biosynthetic pathway strictly according to the sequence of Fig. 17-18. 

CoA labeled with 14C (radioactive carbon) at the carboxyl group, CH3—14C—SCoA, into the citric acid cycle, the 2-oxopentanedioate acid (2-ketoglutarate) formed in the fourth step of the cycle would have all of the 14C in the carboxylate group farthest away from the ketone carbonyl group. For some years, this result was used to argue that citric acid itself could not be an intermediate in the formation of 2-oxopentanedioate. Review Section 19-8 and explain how, in stereospecific enzyme-induced reactions, citric acid could be an intermediate in the formation of 2-oxopentanedioate even if the 14C would not appear equally in both carboxylic carbons of the product. 

See also Citric Acid Cycle, Glyoxylate Cycle Reactions, Stereospecificity of Aconitase, Enzymes of the Citric Acid Cycle, Table 14.1... 

Popjak, G. Stereospecificity of Enzyme Reactions. In Boyer, P. D., ed.. The Enzymes, 3rd ed., Vol. 2, Kinetics and Mechanism. New York Academic Press, 1970. [A review of stereochemical aspects of the citric acid cycle.]... 

The citric acid cycle, also known as the tricarboxylic acid cycle or the Krebs cycle, is the final oxidative pathway for carbohydrates, lipids, and amino acids. It is also a source of precursors for biosynthesis. The authors begin Chapter 17 with a detailed discussion of the reaction mechanisms of the pyruvate dehydrogenase complex, followed by a description of the reactions of the citric acid cycle. This description includes details of mechanism and stereospecificity of some of the reactions, and homologies of the enzymes to other proteins. In the following sections, they describe the stoichiometry of the pathway including the energy yield (ATP and GTP) and then describe control mechanisms. They conclude the chapter with a summary of the biosynthetic roles of the citric acid cycle and its relationship to the glyoxylate cycle found in bacteria and plants. 

Mevalonic acid itself is a product of acetate metabolism. Three molecules of acetate coenzyme A, produced by the citric acid cycle, are used to form mevalonic acid (Scheme 5.1). Two molecules undergo a Claisen condensation via acetyl-CoA-acetyltransferase enzyme [EC 2.3.1.9] to produce acetoacetyl-CoA, and a third is incorporated in a stereospecific aldol addition to the formation of p-hydroxy-p-methylglutaryl-CoA (HMG-CoA) by the aid of HMG-CoA synthase [EC 2.3.3.10]. The first Claisen reaction was found to involve formation of Cys-89 acetyl-5-enzyme reaction intermediate [9]. Then, Cys-378 residue on the active site of the enzyme activates a second molecule of acetyl-CoA to initiate the condensation reaction (Fig. 5.4) [11]. Similarly, in HMG-CoA synthases (S. aureus HMG-CoA synthase), Cysl 11/129 are the crucial residues of covalent attach to acetyl-CoA to produce acetyl-enzyme thioester with the subsequent loss of coenzyme A (Fig. 5.4). Glu79/95 residues are responsible for the enolization of acetyl-enzyme intermediate in order to react with acetoacetyl-CoA, which is bound to His233/264 residues [12]. 

The reaction that uses most of the acetyl-CoA formed by pyruvic acid is the condensation of acetic acid and the keto form of oxaloacetic acid to yield citric acid by forming a carbon-to-carbon bond between the methyl carbon of acetyl-CoA and the carbonyl carbon of oxaloacetate. This reaction is interesting in more than one respect. In addition to the fact that the reaction introduces the oxidation product of fatty acids, carbohydrates, and proteins into the tricarboxylic cycle by converting the 4-carbon chain of oxaloacetic acid into a 6-carbon chain (citric acid), the condensing enzyme presents a fascinating stereospecificity. 

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