The mechanism of action of yeast beta-hydroxy-beta-methylglutaryl-coenzyme A reductase has been investigated through kinetic studies on the oxidation of mevaldate by nicotinamide adeninine dinucleotide phosphate (NADP) in the presence of coenzyme A (CoA) and on the reduction of mevaldate by reduced NADP (NADPH) in the absence of presence of CoA or acetyl-CoA. NADP and mevalonate were also used as product inhibitors of the reduction of mevaldate. In the reduction of mevaldate to mevalonate, coenzyme A and acetyl-CoA decreased the Km for mevaldate 30- and 3-fold, respectively. Both compounds increased the Vmax 1.5-fold. These results suggest that CoA is an allosteric activator for the second reductive step and that it acts by enhancing the binding of mevaldate. The intersecting patterns obtained from initial velocities and the patterns produced by product inhibitions suggest the following features of the mechanism. The binding of substrates and release of products proceeds sequentially in both reductive steps, and is ordered throughout or random with respect to the binding of the beta-hydroxy-beta-methylglutaryl-coenzymeA and the first NADPH. The binding of NADPH enhances the binding of the beta-hydroxy-beta-methylglutaryl portion of the CoA ester and the binding of free mevaldate, whereas the binding of NADP leads to an increased affinity of the enzyme for the hemithioacetal (of mevaldate and CoA) and for mevalonate. Thus, the replacement of NADP by NADPH after the first reductive step promotes the conversion of the hemithioacetal to the free carbonyl form, which is then rapidly reduced. The products, CoA and mevalonic acid, of the second reductive step leave the enzyme before the release of the second NADP. This release of the last product is probably the rate-limiting step for the overall process.
Diurnally varying activity of hepatic jB-hydroxy-fl-methylglutaryl coenzyme A reductase (EC 1.1.1.34) was decreased to very low levels in hypophysectomized rats with no discernable diurnal rhythm retained. Administration of triiodothyronine (100 jg/100 g of body weight) produced a supranormal level of reductase activity, about 3-4 times the highest activity found in normal rats. 0-Hydroxy-#-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) catalyzes the reduction of HMG-CoA to mevalonate, which is the first committed step in the pathway of cholesterol biosynthesis. This reaction has been established as rate-limiting in the over-all conversion of acetate to cholesterol (1, 2). Indeed, changes in HMG-CoA reductase activity accurately reflect changes in the rate of cholesterol synthesis (3, 4). Interestingly, the activity of the reductase undergoes daily changes of as much as 10-fold (5-8). The normal diurnal rise in reductase activity has been shown to be due to an increase in the rate of enzyme synthesis (9). Recently, we have shown (10) that the activity of HMGCoA reductase was stimulated markedly by insulin treatment. We have further demonstrated that reductase activity was reduced to low levels in diabetic rats with only a negligible diurnal rhythm present seven days after the onset of diabetes (11). Administration of insulin restored the activity to nearly normal levels within 2 hr. Other hormones that have been implicated in stimulating cholesterol biosynthesis are thyroid hormones (12, 13) and norepinephrine (14). Guder et al. (13) have shown that hepatic HMG-CoA reductase activity was decreased to less than 50% of normal in hypothyroid rats (produced by 13l1 treatment). A single injection of triiodothyronine (T3) to these hypothyroid rats restored the activity to that of normal animals within 48 hr. In hypophysectomized rats, hepatic cholesterogenesis was reduced to even lower levels (15) than in hypothyroid rats.In this communication we demonstrate that hepatic HMGCoA reductase activity is markedly lowered in hypophysectomized rats with no detectable diurnal rhythm present. Administration of T3 to these animals increases reductase activity to a supranormal level (3-4 times the highest level found in normal rats).
MATERIALS AND METHODS
Materials. iTriiodothyronine (T3) and [3-14C]0-hydroxy-,-methylglutaric acid were purchased from Sigma Chemical and New England Nuclear Corp., respectively. Other chemicals were of reagent grade.[s-'4C ]b-Hydroxy-g-methylglutaric acid was converted to the anhydride by the dicyclohexylcarbodiimide method of Goldfarb and Pitot (16). The coenzyme A ester was prepared from the anhydride as described by Hilz et al. (17) and then purified by descending paper chromatography on Whatman 3 MM with isobutyric acid-concentrated ammonia-0.1 M EDTA (pH 4.5)-H20 (124:5:2:75) as the solvent system. Treatment of Animals. Young adult male albino rats weighing 140-160 g were purchased from Holtzman. Animals were hypophysectomized by Altech Laboratories of Madison, Wisc. The h...
Beta-Hydroxy-beta-methylglutaryl-coenzyme A reductase of yeast has been solubilized by two different methods and then purified approximately 5000-fold. The purified enzyme shows a single precipitin band on immunodiffusion, and it moves as a single band of protein and enzyme activity on gel filtration and diethylaminoethylcellulose column chromatography. It also shows one major band on polyacrylamide gel electrophoresis. The specific activity of the pure enzyme is 18 000 to 22 000 nmol of reduced nicotinamide adenine dinucleotide phosphate oxidized per min per mg of protein. The molecular weights of the enzyme, estimated by gel filtration, and the subunits, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, are 2.6 X 10(5) and 6.0 X 10(4), respectively.
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