Thiol/disulfide exchange between 3-hydroxy-3-methylglutaryl-CoA reductase and glutathione. A thermodynamically facile dithiol oxidation.
In glutathione redox buffers, rat liver, microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase rapidly equilibrates between a reduced, active form and an oxidized, inactive form. At pH 7.0, 37 degrees C, the second order rate constant for inactivation of the reduced enzyme by GSSG is 1700 +/- 200 M-1 min-1, approximately 20-fold faster than the reaction of GSSG with a typical, unhindered thiol of pKa 7.7. High concentrations of GSH or lower concentrations of dithiothreitol restore the activity of the oxidized enzyme. The oxidation of the enzyme by GSSG is only 30-fold slower in the presence of saturating levels of both substrates. The incomplete inhibition of thiol/disulfide exchange by substrates can lead to significant changes in the activity of the enzyme during the assay when glutathione is present. At redox equilibrium, both in the absence and presence of substrates, the activity of the enzyme depends on the quantity [GSH]2/[GSSG], suggesting that the redox transition involves the formation of a protein-SS-protein disulfide. The equilibrium constant for the reaction HMGRred + GSSG in equilibrium HMGRox + 2 GSH is 0.55 +/- 0.07 M in the absence of substrates and 0.20 +/- 0.02 M in the presence of saturating levels of both substrates. Thus, HMG-CoA reductase is very sensitive to dithiol oxidation both kinetically and thermodynamically. Significant changes in the oxidation state and activity of this enzyme could be expected to result from normal changes in the thiol/disulfide oxidation state of the cellular glutathione redox buffer.
Cappel
R E
RE
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Gilbert
H F
HF
D000818:Animals; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D005978:Glutathione / Q000378:metabolism* / Q000494:pharmacology; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013816:Thermodynamics
D004220:Disulfides; D004793:Enzyme Reactivators; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D013438:Sulfhydryl Compounds; D006903:Hydroxymethylglutaryl CoA Reductases; D005978:Glutathione; D004229:Dithiothreitol