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 (Ea), thiol-deficient microsomes contain an inactive, latent enzyme (E1) which can be activated by addition of thiols. Ea 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 greater than glutathione (GSH) greater than cysteine. Ea is inhibited by oxidized glutathione (GSSG). The degree of the inhibition of Ea 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 104 000 by gel filtration chromatography on Sepharose 6B. The reducing agents NaBH4, dithionite and ascorbate failed to activate E1. NaBH4 did not inhibit Ea whereas only partial inhibition was caused by ascorbate and dithionite. Soluble Ea 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 Ea is affected by increasing the affinity of the enzyme to the substrate hydroxymethylglutaryl-CoA.
D000818:Animals; D003545:Cysteine / Q000494:pharmacology; D004226:Dithioerythritol / Q000494:pharmacology; D004789:Enzyme Activation; D005978:Glutathione / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D013438:Sulfhydryl Compounds; D004226:Dithioerythritol; D006903:Hydroxymethylglutaryl CoA Reductases; D005978:Glutathione; D003545:Cysteine