Role of cysteine residues in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A reductase

Tuajuanda C Jordan-Starck, Purdue University

Abstract

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), overexpressed in Escherichia coli (Beach, M. J., and V. W. Rodwell (1989) J. Bacteriol., in press) was purified to electrophoretic homogeneity in 75% yield to a final specific activity of 48 $\mu$mols NAD$\sp+$ reduced/min/mg protein. The overexpressed and Pseudomonas enzymes were virtually indistinguishable kinetically and catalyzed essentially the same four reactions catalyzed by the eukaryotic enzymes. The rates of the four reactions, K$\sb{\rm M}\sp\prime$s for all substrates, and the pH optima for two reactions were measured. No reduced thiol was required. Coenzyme A decreased K$\sb{\rm M}$ for mevaldehyde 12-fold and increased V$\sb{\rm max}$ 2- to 3-fold. Titration with 5,5$\sp\prime$-dithiobis(2-nitrobenzoate) (DTNB) indicated two sulfhydryls per enzyme subunit. Both sulfhydryls remained accessible to DTNB in the presence of mevalonate, NAD$\sp+$, or mevalonate + NAD$\sp+$; only one in the presence of HMG-CoA. N-Ethylmaleimide (NEM) equally inhibited all four reactions. HMG-CoA, but not mevalonate or NAD$\sp+$, afforded protection from NEM inactivation. Methyl methanethiosulfonate (MMTS) completely and irreversibly inactivated the enzyme. Both cysteines, Cys$\sp{156}$ and Cys$\sp{296}$, were replaced with alanines by site-directed mutagenesis. The mutant enzymes, C156A, C296A, and C156/296A were overexpressed in Escherichia coli and purified. The alanine replacements had no significant effect on specific activity or on affinity for any substrate. The mutants catalyzed all four reactions as efficiently as wild-type enzyme. C156A and C156/296A were not inactivated by DTNB, MMTS or NEM. By contrast, C296A was inactivated to the same extent as wild-type enzyme.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry

COinS