Human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase: expression, purification, and characterization of recombinant wild-type and Cys129 mutant enzymes
| Autor: | L. L. Rokosz, P. A. Lachance, Gautam Sanyal, M. A. Cueto, E. A. Butkiewicz, D. A. Boulton, J. D. Hermes |
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| Rok vydání: | 1994 |
| Předmět: |
Hydroxymethylglutaryl-CoA Synthase
Coenzyme A Mutant Molecular Sequence Data Biophysics Biochemistry law.invention Serine chemistry.chemical_compound Lactones Structure-Activity Relationship law Acetyl Coenzyme A Escherichia coli Humans Amino Acid Sequence Cysteine Cloning Molecular Molecular Biology Fluorescent Dyes Gene Library Alanine chemistry.chemical_classification ATP synthase biology Base Sequence Sequence Homology Amino Acid Wild type Sequence Analysis DNA Molecular biology Recombinant Proteins Enzyme chemistry Mutation biology.protein Recombinant DNA Fatty Acids Unsaturated Mutagenesis Site-Directed |
| Zdroj: | Archives of biochemistry and biophysics. 312(1) |
| ISSN: | 0003-9861 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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