Abstract
The feeding of mevinolin plus cholestyramine to rats results in the production of a form of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR-CM) having thiol/disulfide redox properties different from those of 3-hydroxy-3-methylglutaryl-CoA reductase isolated from animals which had been given only cholestyramine (HMGR-C). The second-order rate constant for the inactivation of HMGR-CM by GSSG is 7-fold slower than for HMGR-C, while the second-order rate constant for the reactivation of oxidized enzyme by GSH is 100-fold slower. However, in the presence of saturating concentrations of both substrates, the rate constants for thiol/disulfide exchange are similar for both forms of the enzyme. HMGR-CM behaves as if a protein-glutathione mixed disulfide having a Kox of 27 +/- 4 is formed at equilibrium. In contrast, HMGR-C has previously been shown to form a protein-protein disulfide (Cappel, R. E., and Gilbert, H. F. (1988) J. Biol. Chem. 263, 12204-12212). Both forms of the enzyme are more difficult to oxidize thermodynamically in the presence of saturating levels of both substrates. For HMGR-CM, NADPH alone has no effect on the equilibrium constant for oxidation, but hydroxymethylglutaryl-CoA alone makes the enzyme approximately twice as difficult to oxidize. Under physiological conditions, HMGR-CM is thermodynamically more difficult to oxidize than HMGR-C. HMGR-C can be converted to HMGR-CM by in vitro treatment with mevinolinate. A direct or indirect interaction of mevinolin with HMGR-C results in some persistent, as yet undefined, structural alteration which inhibits the formation of a protein-SS-protein disulfide upon oxidation by glutathione disulfide.
Highlights
Methylglutaryl-CoAreductase (HMGR-CM) having thiol/disulfide redox properties different from thoosfe 3-hydroxy-3-methylglutaryl-CoAreductaseisolated from animals which had been given only cholestyramine (HMGR-C)
Under physiological con- tyramine and mevinolin (HMGR-CM) exhibitsdifferent ditions, HMGR-CM is thermodynamically more diffi- properties than theenzyme isolated from the livers of rats fed cult to oxidize than HMGR-C
A direct or indirect interactioonf mevinolin with HMGR-C results in some persistent, as yet undefined, structural alteration which inhibits the formation of a protein-SS-protein disulfide upon oxidation by glutathione disulfide
Summary
Control in which 22 mM D T T was substituted for GSH, was used to define the Vmax.For the DTT control, the rate of RESULTS. The 40-min assay period; when GSSG was present, Redox Equilibriumin the Presence of One or Both Subthe rateof lactone productiondecreased with time, indicating strates-The KO, for rat liver HMGR-CMunderturnover that theenzyme was being inactivated during the assay(Fig. conditions was determined in a manner similar to that de-. The enzyme was preincubated with a series of loss of enzymatic activity under these conditions was deter- glutathione redox buffers containing various [GSH]/[GSSG]. Occur under turnover conditions.Addition of oxidatively in- fold dilution of the preincubate), the assay twimase increased activated enzyme toanassaymixturecontainingvarious to 60 min, and the concentration of enzyme in the preincuconcentrations of GSH resulted in a significant increase in bation had to be lowered to ensure that HMG-CoA was not the rateof lactone formation with time(Fig. 4, Miniprint). The presenceof NADPH duringredox equilibration has no significant effect on theKO,,and HMG-CoA alone only makes the enzyme about 2-fold more difficult to oxidize (lower KO%)
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