Kinetic Analysis of the Reaction Catalyzed by Rat-Liver 3-Hydroxy-3-methylglutaryl-coenzyme-A Reductase Using Two Specific Inhibitors.

The mechanism of action of 3-hydroxy-3-methylglutaryl-CoA reductase solubilized from rat liver microsomes has been investigated. In the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate, an overall initial velocity study gave a linear intersecting pattern when both 3-hydroxy-3- methylglutaryi-CoA and NADPH were variable substrates. Adenosine 2′-monophospho-5′-diphos- phoribose, which was found to be a reversible inhibitor of reductase, inhibited the enzyme competitively with respect to NADPH, and uncompetitively with respect to 3-hydroxy-3-methylglutaryl-CoA. On the other hand, the inhibition of reductase by ML-236B (sodium salt), a specific reversible inhibitor of the enzyme isolated from the culture of a Penicillium (whose structure is given in the paper) is competitive with respect to 3-hydroxy-3-methylglutaryl-CoA and noncompetitive with respect to NADPH [Endo et al. (1976) FEBS Lett. 72, 323-326]. The reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate was subject to the substrate inhibition by NADPH attributed to the formation of a productive enzyme-NADPH complex. These results indicate that the two substrates bind to the enzyme effectively in an ordered manner; reductase first interacts with 3-hydroxy-3-methylglutaryl-CoA to make a binary complex, which, in turn, forms a ternary complex with one molecule of NADPH. Considered together with the results of product inhibition study, and assuming a hemithioacetal of mevaldate and CoA is an intermediate of the reductase reaction, a bi-uni-uni-ter-ping- pong mechanism is proposed as a model of the overall reaction. [ABSTRACT FROM AUTHOR]