AVIAN CYTOSOLIC 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE - EVALUATION OF THE ROLE OF CYSTEINES IN REACTION CHEMISTRY

The pH dependence of avian cytosolic HMG-CoA synthase activity is fit by a titration curve with a pK = 8.6. The observation of optimal activ ity at alkaline pH and the insensitivity of pK to divalent cation conc entration suggest that the pK reflects ionization of an amino-acid sid e chain (e.g., cysteinyl sulfhydryl) rather than substrate enolization . Upon reaction of 3-chloropropionyl-CoA with HMG-CoA synthase C129S, an enzyme variant lacking the sulfhydryl group normally targeted by th is mechanism-based inhibitor, stoichiometric modification occurs. Amin o-acid analysis indicates that cysteine is the principal target in C12 9S enzyme, demonstrating the presence of a second reactive cysteine wi thin this enzyme. To test whether another cysteine functions in reacti on chemistry, conserved cysteines were identified by sequence homology analysis. Five cysteine residues (C59, C69, C224, C232, C268), invari ant in the nine sequences available for various eukaryotic HMG-CoA syn thase isozymes, were individually replaced by alanine in a series of m utant enzymes. Kinetic analyses of the isolated mutant HMG-CoA synthas es indicate that none of these is crucial to the chemistry that result s in production of HMG-CoA. These results further distinguish the HMG- CoA synthase reaction from the related condensation of acyl-CoA substr ates catalyzed by beta-ketothiolase.