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

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 105. 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. Km values are 0.050 mm (dl-HMG-CoA), 0.032 mm (NADH), 0.30 mm (mevalonate), 0.27 mm (NAD+), and 0.039 mm (CoA). Although HMG is a competitive inhibitor both of mevalonate oxidation (Ki = 0.22 mm) and of HMG-CoA reduction (Ki = 3.9 mm), the former reaction is more effectively inhibited (Ki:Km = 0.73) than is the latter (Ki:Km = 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, Km values are 0.61 mm (dl-mevaldate), 0.06 mm (NAD+), and 0.046 mm (CoA). HMG is an effective inhibitor competitive with mevaldate (Ki = 1.3 mm; Ki:Km = 2.1). For Reaction 3, Km values are 9 mm (dl-mevaldate) and 0.36 mm (NADH); HMG is an effective inhibitor competitive with mevaldate (Ki = 2.3 mm; Ki:Km = 0.25).