Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase. Evidence for thiol-dependent allosteric modulation of enzyme activity.
Rat liver microsomes devoid of free thiols were prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer in the presence of 30 microM leupeptin. The activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by GSH and dithiothreitol (DTT) in these microsomes was studied and compared to the activation by these thiols of enzyme that was solubilized by freeze-thawing. An increase of Vmax was observed, for the two enzyme preparations, with increasing concentrations of the two activating thiols. Reactions of GSH-activated microsomal enzyme with increasing concentrations of NADPH show sigmoidal kinetics with a Hill coefficient of 2.01 +/- 0.07 at 2-4 mM GSH. Increase of the activating GSH concentrations resulted in a gradual change towards Michaelis-Menten kinetics, and a Hill coefficient of 1.08 +/- 0.03 for NADPH was calculated at 25 mM GSH. Activation of the microsomal enzyme by DTT yielded similar results except that a Hill coefficient of 1.1 was observed already at 2.5 mM DTT. Normal Michaelis-Menten kinetics were observed for HMG-CoA at all GSH concentrations. Solubilization of HMG-CoA reductase by the widely used freeze-thaw procedure abolished the cooperative pattern, and normal Michaelis-Menten kinetics with a Hill coefficient of 1.0 was observed regardless of GSH concentration. These results are compatible with a model in which HMG-CoA reductase activity is GSH-dependent, allosterically modulated under physiological hepatic conditions. In addition, the widely used assay conditions, using high DTT concentrations or employment of highly purified soluble enzyme precluded the observation of sigmoidal kinetics and the suggested model.
D000818:Animals; D004229:Dithiothreitol / Q000494:pharmacology*; D004789:Enzyme Activation; D005978:Glutathione / Q000494:pharmacology*; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D009249:NADP; D006903:Hydroxymethylglutaryl CoA Reductases; D005978:Glutathione; D004229:Dithiothreitol