Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Characterization and chemical modification.
Pseudomonas mevalonii (formerly designated Pseudomonas sp. M (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001; Gill, J. F., Jr., Beach, M.J., and Rodwell, V. W. (1985) J. Biol. Chem. 260, 9393-9398] 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), overexpressed in Escherichia coli (1), has been purified to electrophoretic homogeneity in 75% yield (final specific activity 48 mumols of NAD+ reduced per min/mg protein). The enzyme catalyzes its normal catabolic reaction (mevalonate + 2 NAD+ + CoASH----HMG-CoA + 2NADH + 2H+), and two half-reactions which involve mevaldehyde, the postulated intermediate in the aforementioned reactions and mevaldehyde + NADH + H+----mevalonate + NAD+). The rates of all four reactions and the Michaelis constants for all substrates were measured. Coenzyme A decreased the KM for mevaldehyde reduction 12-fold and stimulated VMAX 2-3 fold. CoASH thus may remain bound throughout the catalytic cycle. Dithiothreitol and analogs of CoASH were tested for their ability to reproduce the CoASH stimulation. Pantetheine, but not dithiothreitol, pantothenate, or desulfo-CoA mimicked CoASH stimulation. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) indicated two sulfhydryl groups per subunit. Both groups remained accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of mevalonate and/or NAD+ but only one group in the presence of HMG-CoA. N-Ethylmaleimide inhibited all the aforementioned reactions. HMG-CoA, but not mevalonate, afforded protection completely and irreversibly inactivated the enzyme. The reactive sulfhydryl group thus may not be a catalytic residue, but may be involved in a conformational change.
Jordan-Starck
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Rodwell
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D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics
D004228:Dithionitrobenzoic Acid; D006903:Hydroxymethylglutaryl CoA Reductases; D005033:Ethylmaleimide