Citrate synthase enzymic 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 3. Mitochondrial fatty acid oxidation. Long-chain fatty acids are converted to their CoA-esters as described in the text, and their fatty-acyl-groups transferred to CoA in the matrix by the concerted action of CPT 1, the acylcarnitine/carnitine exchange carrier and CPT (A) as described in the text. Medium-chain and short-chain fatty acids (Cg or less) diffuse directly into the matrix where they are converted to their acyl-CoA esters by a acyl-CoA synthase. The mechanism of p-oxidation is shown below (B). Each cycle of P-oxidation removes -CH2-CH2- as an acetyl unit until the fatty acids are completely converted to acetyl-CoA. The enzymes catalyzing each stage of P-oxidation have different but overlapping specificities. In muscle mitochondria, most acetyl-CoA is oxidized to CO2 and H2O by the citrate cycle (Figure 4) some is converted to acylcamitine by carnitine acetyltransferase (associated with the inner face of the inner membrane) and exported from the matrix. Some acetyl-CoA (if in excess) is hydrolyzed to acetate and CoASH by acetyl-CoA hydrolase in the matrix. Enzymes ...

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. 

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... 

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). 

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. 

Long chain acyl-CoA esters cause a competitive inhibition of acetyl-CoA-carboxylase. The elevated levels of long chain fatty acid Co-A esters in diabetes and on starvation may be the basis of the keto-acidosis (Bobtz et al. 1963). Citrate synthase (condensing enzyme) is also inhibited by long chaiii acyl-CoA esters (Wieland et al. 1963 a, b). It appears therefore that the product of fatty acid synthesis is able to regulate the synthesis by means of a feed back mechanism. 

During the formation of carboxylic acid like lA, there will be shuttling of metabolites within the intracellular compartments, having the capability to utilize the enzymes of the respective compartments. Jaklitsch et al. (1991) reported that the CadA, which is e key enzyme for the biosynthesis of lA, is located in cytosol. Otiier important enzymes, such as citrate synthase and aconitase, are found in the mitochondria, but some residual level of these enzymes are also found in the cytosolic fraction. The depicted mechanism is that the ds-aconitate is transported to cytosol assisted by the malate-citrate antiporter. The biosynthetic pathway of LA in the citric acid cycle is illustrated in Fig. 10.5. 

Glucagon decreases cholesterol synthesis in isolated hepatocytes [131,132] apparently because it reduces the fraction of hydroxymethylglutaryl-CoA reductase in the active form [131,132], This is due to an increase in reductase kinase activity [133], However, there is no evidence that cAMP-dependent protein kinase phos-phorylates either the reductase, reductase kinase or reductase kinase kinase [134], It has been proposed that the phosphorylation state of these enzymes is indirectly controlled through changes in the activity of protein phosphatase I [132,134], This phosphatase can dephosphorylate and activate the reductase [134,135] and its activity can be controlled by a heat stable inhibitor (inhibitor 1), the activity of which is increased by cAMP-dependent phosphorylation [136,137], Since the phosphorylated forms of acetyl-CoA carboxylase, ATP-citrate lyase, pyruvate kinase, phos-phorylase, phosphorylase kinase and glycogen synthase are also substrates for protein phosphatase I [135], this mechanism could also contribute to their phosphorylation by glucagon. 

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