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‐methylglutaryl‐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 , 323–3261. 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‐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.