Citrate synthase reaction mechanism

FIGURE 20.5 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nncieophiiic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis. 

The final application considered in this chapter is chosen to illustrate the application of a QM-MM study of an enzyme reaction that employs an ab initio Hamiltonian in the quantum region [67]. Because of the computational intensity of such calculations there are currently very few examples in the literahire of QM-MM shidies that use a quanhim mechanical technique that is more sopliisticated than a semiempirical method. MuUiolland et al. [67] recently reported a study of part of the reaction catalyzed by citrate synthase (CS) in wliich the quanhim region is treated by Hartree-Fock and MP2 methods [10,51],... 

Scheme 4.7 Abbreviated reaction mechanism of citrate synthase.

The first step has a AG0 of —0.05 kcal/mole, which is close to zero it does not occur to any great extent unless the concentrations of acetyl-coenzyme A (acetyl-CoA) and oxaloacetate are greater than the concentration of citryl-CoA. The second step, however, has a highly favorable AG0 of — 8.4 kcal/mole. When the two steps are combined, AG0 for the overall reaction is about —8.3 kcal/mole, and the equilibrium constant lies far in the forward direction. These two reactions are catalyzed by the enzyme citrate synthase, by a mechanism that ensures that they always occur together. 

As an example to illustrate analysis of kinetic data to characterize the mechanism of a real enzyme, here we apply the general compulsory-order ternary mechanism introduced above to citrate synthase to determine kinetic parameters for several isoforms of this enzyme and to elucidate the mechanisms behind inhibition by products and other species not part of the overall chemical reaction. 

M. Karpusas, B. Branchaud, and S.J. Remington. 1990. Proposed mechanism for the condensation reaction of citrate synthase 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A. Biochemistry 29 2213-2219. (PubMed)... 

The central role of thiol ester compounds in nutritional biochemistry is illustrated by considering some features of the mechanisms of the reactions catalyzed by citrate synthase and acctyl-CoA carboxylase. A brief background on the properties of the thiol ester bond is first presented. This tnaterial has been simplified and is intended only to clarify why the reactions employ thiol esters of carboxylic acids, rather than oxygen esters or the free, unesterified carboxylic acid. 

However, these transporters do not contain any ABC motifs and transport monocarboxylic acids via a proton-coupled reaction. Acetic acid resistance in A. acetii is, therefore, conferred by at least two mechanisms assimilation of the weak acid by enzymes, such as citrate synthase or aconitase and export of acetic acid by ABC transporter. Both mechanisms are implicated in reducing intracellular acetic acid concentration (Nakano, Fukaya, and Horinouchi, 2006). 

Mechanism The Mechanism of Citrate Synthase Prevents Undesirable Reactions... 

Scheme 10 General reaction mechanism of the CoA-dependent Claisen-type condensing enzymes, malate synthase, a-isopropylmalate synthase, citrate synthase, and homocitrate synthase.

There are, however, clear stereomechanistic differences between these two classes of enzyme-catalyzed reactions. The Claisen-type condensations uniformly involve inversion of configuration at the a-carbon of the esteratic substrate, involving C-C bond formation at either the re or the si face of the ketonic or aldehydic substrate (Table VII) (196-211). Moreover, neither Schiff bases nor metal ions have been directly implicated in the catalytic mechanisms of these enzymes. Unlike the aldolases, these enzymes do not catalyze rapid enolization of the nucleophilic substrate in the absence of the second substrate. Inversion of configuration suggests that at least two catalytic groups, perhaps operating in concert, facilitate C-C bond formation. Physicochemical measurements on citrate synthase are consistent with this interpretation of inversion of configuration. 

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