Citrate synthase mechanism

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],... 

Figure 5 A suggested mechanism for the enolization of acetyl-CoA by the enzyme citrate synthase (CS). The keto, enolate, and enol forms of the substrate are shown.

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. 

Figure 26.10 MECHANISM Mechanism of the addition of acetyl CoA to oxaloacetate to give (S)-citryl CoA, catalyzed by citrate synthase.

Citrate, prochirality of, 1156 Citrate synthase, active site of, 1046 function of, 1045 mechanism of action of, 1043, 1047... 

Scheme 4.7 Abbreviated reaction mechanism of citrate synthase.

Remington, S.J. (1992) Structure and mechanism of citrate synthase. Curr. Tap. Cell. Regul. 33, 209-229. 

Weigand, G. Remington, S.J. (1986) Citrate synthase structure, control, and mechanism. Annu. Rev. Biophys. Biophys. Chem. 15, 97-117. 

Expands and updates the presentation of the mechanism for pyruvate carboxylase. Adds coverage of the mechanisms of isocitrate dehydrogenase and 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. 

Fig. 5 Putative mechanism of citrate synthase.4 A low-barrier hydrogen bond helps to stabilize the enol and tetrahedral intermediates.

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. 

Kinetic parameter values for citrate synthase for several isoforms of the model may be estimated for the general analysis of the compulsory-order ternary mechanism outlined in Section 4.4.3.1 based on observed data. The parameter estimates for citrate synthase obtained from rat kidney, rat liver, and bovine heart are listed in Table 4.2. 

Figure 17.11. Mechanism of Synthesis of Citryl CoA hy Citrate Synthase. The condensation of oxaloacetate and acetyl CoA proceeds through an enol intermediate. The subsequent hydrolysis of citryl CoA yields citrate and CoA.

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)... 

For some enzymes, for example many metalloenzymes, a semiempirical QM/MM treatment is inadequate due to the limitations of the semiempirical methods. In such situations, a more sophisticated level of QM treatment (such as ab initio molecular orbital or density-functional theory) may well be required. An recent example of the application of ab initio QM/MM techniques to an enzyme mechanism is a study of acetyl-CoA enolization in citrate synthase... 

The thiol ester linkage has been mentioned at various points in this text, most frequently in the material on energy metabolism. This bond occurs when a carboxylic acid is conjugated with a sulfhydryl group, such as the one on coenzyme A. The thiol ester linkage is an important concern to those studying the mechanisms of action of acetyi-CoA carboxylase, citrate synthase, piopionyl-CoA carboxylase, and HMG-CoA synthase. 

---