Purification and Properties of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Pseudomonas

• Published in: The Journal of biological chemistry Vol. 245; no. 15; pp. 3755 - 3762
• Main Authors: Bensch, W R, Rodwell, V W
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.08.1970
• Subjects: Alcohol Oxidoreductases - antagonists & inhibitors; Alcohol Oxidoreductases - isolation & purification; Aldehydes; Carbon Isotopes; Coenzyme A; Electrophoresis, Disc; Esterases; Glutarates; Hydrogen-Ion Concentration; Kinetics; Lyases; Mevalonic Acid; Molecular Weight; NAD; Pseudomonas - enzymology; Valerates; Vibration
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)62916-3

An inducible 3-hydroxy-3-methylglutaryl (HMG) coenzyme A reductase (mevalonate:NAD + oxidoreductase (acylating CoA), EC 1.1.1.-) has been purified 21-fold from soluble extracts of Pseudomonas to an apparently homogeneous state as judged by disc gel electrophoresis. The molecular weight estimated by gel electrophoresis is 2.6 to 2.8 x 10 5 . The purified enzyme is free of HMG-CoA lyase (3-hydroxy-3-methylglutaryl-CoA acetoacetate lyase, EC 4.1.3.4) or HMG-CoA hydrolase (3-hydroxy-3-methylglutaryl-CoA hydrolase, EC 3.1.2.5) activities, and catalyzes the following reactions: HMG-CoA + 2NADH + 2H + ⇄ mevalonate + 2NAD + + CoA (1) Mevaldate + NAD + + CoA → HMG-CoA + NADH + H + (2) Mevaldate + NADH + H + → mevalonate + NAD + (3) Parallel enrichment of all three activities is observed throughout purification, suggesting that all are properties of a single protein. Reaction 1 is reversible, but mevalonate formation is strongly favored. Mevaldate is not a free intermediate either in HMG-CoA reduction or mevalonate oxidation. The pH optimum for mevalonate oxidation occurs at pH 9.2 to 9.6. K m values are 0.050 m m ( dl -HMG-CoA), 0.032 m m (NADH), 0.30 m m (mevalonate), 0.27 m m (NAD + ), and 0.039 m m (CoA). Although HMG is a competitive inhibitor both of mevalonate oxidation ( K i = 0.22 m m ) and of HMG-CoA reduction ( K i = 3.9 m m ), the former reaction is more effectively inhibited ( K i :K m = 0.73) than is the latter ( K i :K m = 79). Double reciprocal plots of data obtained by varying both HMG-CoA and NADH concentration given lines intersecting on the negative abscissa, with no evidence of concave curvature. HMG-CoA reduction thus appears to proceed by a mechanism different from that proposed for yeast HMG-CoA reductase by Kirtley and Rudney ( Biochemistry , 6, 230 (1967)). Reaction 3 yields similar double reciprocal plots. For Reaction 2, K m values are 0.61 m m ( dl -mevaldate), 0.06 m m (NAD + ), and 0.046 m m (CoA). HMG is an effective inhibitor competitive with mevaldate ( K i = 1.3 m m ; K i :K m = 2.1). For Reaction 3, K m values are 9 m m ( dl -mevaldate) and 0.36 m m (NADH); HMG is an effective inhibitor competitive with mevaldate ( K i = 2.3 m m ; K i :K m = 0.25).