Kinetic analysis of the individual reductive steps catalyzed by β-hydroxy-β-methylglutaryl-coenzyme A reductase obtained from yeast

Abstract

The mechanism of action of yeast .beta.-hydroxy-.beta.-methylglutaryl-coenzyme A reductase was investigated through kinetic studies on the oxidation of mevaldate by NADP in the presence of CoA and on the reduction of mevaldate by reduced NADP (NADPH) in the absence or presence of CoA or acetyl-CoA. NADP and mevalonate were also used as product inhibitors of the reduction of mevaldate. In the reduction of mevaldate to mevalonate, CoA and acetyl-CoA decreased the Km for mevaldate 30- and 3-fold, respectively. Both compounds increased the Vmax 1.5-fold. Apparently CoA is an allosteric activator for the 2nd reductive step and it acts by enhancing the binding of mevaldate. The intersecting patterns obtained from initial velocities and the patterns produced by product inhibitions suggest the following features of the mechanism. The binding of substrates and release of products proceeds sequentially in both reductive steps, and is ordered throughout or random with respect to the binding of the .beta.-hydroxy-.beta.-methylglutaryl-coenzyme A and the 1st NADPH. The binding of NADPH enhances the binding of the .beta.-hydroxy-.beta.-methylglutaryl portion of the CoA ester and the binding of free mevaldate, whereas the binding of NADP leads to an increased affinity of the enzyme for the hemithioacetal (of mevaldate and CoA) and for mevalonate. The replacement of NADP by NADPH after the 1st reductive step promotes the conversion of the hemithioacetal to the free carbonyl form, which is then rapidly reduced. The products, CoA and mevalonic acid, of the 2nd reductive step leave the enzyme before the release of the 2nd NADP. This release of the last product is probably the rate-limiting step for the overall process.