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

Abstract

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-methylglutaryl-CoA and NADPH were variable substrates. Adenosine 2'-monophospho-5'-diphosphoribose, 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 FEBS Lett. 72, 323 -3261. 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-methylglutarylCoA 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-pingpong mechanism is proposed as a model of the overall reaction.3-Hydroxy-3-methylglutaryl-CoA reductase is an enzyme of current interest because of its regulatory role in cholesterogenesis [l, 21. The enzyme catalyzes the two-step reduction of 3-hydroxy-3-methylglutarylCoA to mevalonate in the presence of NADPH. 3-Hydroxy-3-methylglutaryl-Previous conclusions obtained from kinetic analyses with the reductase of bakers' yeast are contradictory [3,4]. Thus, Kirtley and Rudney first proposed a penta-uni-bi-ping-pong mechanism on the basis of an initial velocity pattern of parallel lines and product inhibition studies [3]. On the other hand, Qureshi et al. proposed a mechanism ordered throughout or random with respect to the binding of 3-hydroxy-3-methylglutaryl-CoA and the first molecule of NADPH by the analysis of the conversion of mevaldate to 3-hydroxy-3-methylglutaryl-CoA and mevalonate [4]. -.Ahhrroiation. CAMP, adenosine 3' : 5'-monophosphate. Enq-JrnP. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase or mevalonate: NADP oxidoreductase (EC 1.1.1.34).Further, little evidence has been reported on the reaction mechanism of the enzyme of animal origin [5,6].A previous report from this laboratory [7] has shown that the fungal metabolite ML-236B (Fig. 1) is a competitive inhibitor of the reductase. In the present experiments, binding characteristics of substrates to rat liver 3-hydroxy-3-methylglutaryl-CoA reductase was studied with the use of ML-236B and adenosine 2'-monophospho-5'-diphosphoribose, another potent inhibitor of ...

Chemistry, biochemistry, and pharmacology of HMG-CoA reductase inhibitors

“…The inhibition of HMG-CoA reductase by compactin is reversible and is competitive with the substrate HMG-CoA; the inhibitor constant Ki is around 10-9M [6,26]. These data prompted us to notice that the ring-opened structure of the 6-1actone of compactin resembles that of the HMG portion of HMG-CoA.…”

Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent.

“…Recently, Endo and his associates isolated and characterized a metabolite from cultures of Penicillium citrinum that was shown to be an extremely potent competitive inhibitor of HMG-CoA reductase (5)(6)(7)(8). This compound, designated ML-236B, is identical to one isolated from P. brevicompactum as an antifungal agent, which was named compactin (9).…”

“…Mevinolin (Fig. 1, structure 1) is a member in the family of 8 substituted hexahydronaphthalene lactones, which interfere with the biosynthesis of mevalonic acid by inhibition of HMG-CoA reductase. This family of natural products includes obythe nuclear variants of compactin (ML-236B) that lack the 2-rationsk methyl-l-oxobutyl and 2-methyl-1-oxobutoxy groups (ML-236A and C, respectively).…”

Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699.

“…The fungal metabolites lovastatin and compactin are specific competitive inhibitors of the enzyme 3-hydroxy-3-rnethylglutaryl coenzyme A (HMG-CoA) reductase (1)(2)(3). Administration of these compounds to cultured animal cells (4), to intact animals in vivo (1,5), and to humans (6,7) resulted in an overall decrease in the synthesis of cholesterol.…”