HUMAN CYTOPLASMIC 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A SYNTHASE - EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT WILD-TYPE AND CYS(129) MUTANT ENZYMES

A cDNA for the human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5) was subcloned and expressed from a T7 -based vector in Escherichia coil. The over-produced enzyme was purifi ed using a three-step protocol that generated 20 to 30 mg protein/lite r cell culture. The physical and catalytic properties of the recombina nt synthase are similar to those reported for the nonrecombinant enzym es from chicken liver [Clinkenbeard ct al. (1975a) J. Biol. Chem. 250, 3124-3135] and rat liver [Mehrabian et al. (1986) J. Biol. Chem. 261, 16249-16255]. Mutation of Cys(129) to serine or alanine destroys HMG- CoA synthase activity by disrupting the first catalytic step in HMG-Co A synthesis, enzyme acetylation by acetyl coenzyme A. Furthermore, unl ike the wild-type enzyme, neither mutant was capable of covalent modif ication by the p-lactone inhibitor, L-659,699 [Greenspan et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7488-7492]. Kinetic analysis of the in hibition by L-659,699 revealed that this compound is a potent inhibito r of the recombinant human synthase, with an inhibition constant of 53 .7 nM and an inactivation rate constant of 1.06 min(-1). (C) 1994 Acad emic Press, Inc.