Human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase: expression, purification, and characterization of recombinant wild-type and Cys129 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 coli. The over-produced enzyme was purified using a three-step protocol that generated 20 to 30 mg protein/liter cell culture. The...

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Bibliographic Details
Published in:	Archives of biochemistry and biophysics Vol. 312; no. 1; p. 1
Main Authors:	Rokosz, L L, Boulton, D A, Butkiewicz, E A, Sanyal, G, Cueto, M A, Lachance, P A, Hermes, J D
Format:	Journal Article
Language:	English
Published:	United States 01-07-1994
Subjects:	Acetyl Coenzyme A - metabolism Amino Acid Sequence Base Sequence Cloning, Molecular Cysteine - genetics Escherichia coli - genetics Fatty Acids, Unsaturated - chemistry Fatty Acids, Unsaturated - pharmacology Fluorescent Dyes - chemistry Fluorescent Dyes - pharmacology Gene Library Humans Hydroxymethylglutaryl-CoA Synthase - antagonists & inhibitors Hydroxymethylglutaryl-CoA Synthase - genetics Hydroxymethylglutaryl-CoA Synthase - isolation & purification Hydroxymethylglutaryl-CoA Synthase - metabolism Lactones - chemistry Lactones - pharmacology Molecular Sequence Data Mutagenesis, Site-Directed Mutation Recombinant Proteins - metabolism Sequence Analysis, DNA Sequence Homology, Amino Acid Structure-Activity Relationship
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Summary:	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 coli. The over-produced enzyme was purified using a three-step protocol that generated 20 to 30 mg protein/liter cell culture. The physical and catalytic properties of the recombinant synthase are similar to those reported for the nonrecombinant enzymes from chicken liver [Clinkenbeard et al. (1975a) J. Biol. Chem. 250, 3124-3135] and rat liver [Mehrabian et al. (1986) J. Biol. Chem. 261, 16249-16255]. Mutation of Cys129 to serine or alanine destroys HMG-CoA synthase activity by disrupting the first catalytic step in HMG-CoA synthesis, enzyme acetylation by acetyl coenzyme A. Furthermore, unlike the wild-type enzyme, neither mutant was capable of covalent modification by the beta-lactone inhibitor, L-659,699 [Greenspan et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7488-7492]. Kinetic analysis of the inhibition by L-659,699 revealed that this compound is a potent inhibitor of the recombinant human synthase, with an inhibition constant of 53.7 nM and an inactivation rate constant of 1.06 min-1.
ISSN:	0003-9861
DOI:	10.1006/abbi.1994.1273