Human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase: expression, purification, and characterization of recombinant wild-type and Cys129 mutant enzymes

Arch Biochem Biophys. 1994 Jul;312(1):1-13. doi: 10.1006/abbi.1994.1273.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • 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

Substances

  • Fatty Acids, Unsaturated
  • Fluorescent Dyes
  • Lactones
  • Recombinant Proteins
  • antibiotic 1233A
  • Acetyl Coenzyme A
  • Hydroxymethylglutaryl-CoA Synthase
  • Cysteine

Associated data

  • GENBANK/L25798