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 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.
Rokosz
L L
LL
Department of Biophysical Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065-0900.
Boulton
D A
DA
Butkiewicz
E A
EA
Sanyal
G
G
Cueto
M A
MA
Lachance
P A
PA
Hermes
J D
JD
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D015723:Gene Library; D006801:Humans; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007783:Lactones / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D005231:Fatty Acids, Unsaturated; D005456:Fluorescent Dyes; D007783:Lactones; D011994:Recombinant Proteins; C054282:antibiotic 1233A; D000105:Acetyl Coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase; D003545:Cysteine