Thiol-disulfide-dependent interconversion of active and latent forms of rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme a reductase

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase, EC 1.1.1.34) in preparations of thiol-deficient rat liver microsomes and microsomes containing thiols have been compared. Unlike microsomes containing thiols, which possess an active hydroxymethylglutaryl-CoA reductase (E_a), thiol-deficient microsomes contain an inactive, latent enzyme (E₁) which can be activated by addition of thiols. E_a can be converted to E₁ by dialysis. The maximal degree of activation of E₁ depends on the activating thiol with the order of effectiveness: dithioerythritol = dithiothreitol > glutathione (GSH) > cysteine. E_a is inhibited by oxidized glutathione (GSSG). The degree of the inhibition of E_a by GSSG is proportional to the ratio GSSG/thiol in the reaction. E₁ was solubilized from microsomes and purified. Its molecular weight is estimated to be 104000 by gel filtration chromatography on Sepharose 6B. The reducing agents NaBH₄, dithionite and ascorbate failed to activate E₁. NaBH₄ did not inhibit E_a whereas only partial inhibition was caused by ascorbate and dithionite. Soluble E_a binds to both blue dextran/Sepharose 4B and agarose/hexane-3-hydroxy-3-methylglutaryl Coenzyme A affinity resins at low-salt concentrations. By contrast, soluble E₁ did not bind to agarose/hexane-hydroxymethylglutaryl-CoA whereas quantitative binding of E₁ to blue dextran/Sepharose 4B was still observed at low salt concentrations. These results indicate that thiols are necessary cofactors for hydroxymethylglutaryl-CoA reductase reaction. Their effect on the activation of E₁ is not caused by change in the state of aggregation of the enzyme. Rather, the reversible change of the enzyme from E₁ to E_a is affected by increasing the affinity of the enzyme to the substrate hydroxymethylglutaryl-CoA.