Evidence for catalytic site cysteine and histidine by chemical modification of beta-hydroxy-beta-methylglutaryl-coenzyme A reductase.

S-(4-Bromo-2,3-dioxobutyl)-coenzyme A inactivates both yeast and rat liver beta-hydroxy-beta-methylglutaryl-coenzyme A reductase. The inactivation is irreversible, complete in 15 s, and proportional to the concentration of the reagent. beta-Hydroxy-beta-methylglutaryl-CoA provides protection against inactivation, whereas NADPH does not. Inactivation is attributed to reaction with an essential cysteine at the beta-hydroxy-beta-methylglutaryl-CoA binding site. Experiments with other active site-directed reagents confirm the involvement of a cysteine and support the presence of an active-site histidine, but rule out the participation of arginine or serine.

Antibody preference for the catalytically active form of beta-hydroxy-beta-methylglutaryl coenzyme A reductase.

The catalytically inactivating subset within rabbit serum polyclonal antibody to the solubilized, purified 55,000 to 60,000 dalton active fragment of rat liver microsomal beta-hydroxy-beta-methylglutaryl coenzyme A reductase immunoinactivates this enzyme with little or no diminution of effect by enzyme catalytically inactivated by incubation of microsomes with ATP,Mg++. Reactivation of inactive enzyme with ethanol-treated rat liver phosphatase restores antibody affinity showing that the catalytically inactivating subset of antibody exhibits marked or complete affinity for the active enzyme over the ATP,Mg++- inactivated form. This means that immunoinactivation using this antibody is not a valid way of measuring changes in the specific activity of the enzyme via phosphorylation-dephosphorylation. Preference for the active enzyme has not been obvious because when different amounts of enzyme activity are used in immunotitrations of samples of low activity, apparent differences in specific activity are observed when none actually exist. If precautions are not taken, results are obtained supporting phosphorylation by using an antibody that is not capable of distinguishing it.

Regulation of short-term changes in hepatic beta-hydroxy-beta-methylglutaryl-CoA reductase activity.

Immunotitrations of rat liver hydroxymethylglutaryl-CoA (HOMeGlt-CoA) reductase activity were performed before and after short-term changes in the nutritional or hormonal state of the animals. Changes in enzyme activity (increase or decrease) within 1 h following cholesterol feeding or glucagon or mevalonolactone administration to normal rats, or insulin administration to diabetic rats were accompanied by no change in the specific activity of the enzyme, as determined from the quantity of enzyme activity inactivated by a fixed quantity of antibody. These results support the conclusion that the loss in enzyme activity was due to conversion of the enzyme to immuno-unreactive products. In agreement with this conclusion the enzyme activity lost after these short-term physiological changes was not restorable by phosphoprotein phosphatase action. On the other hand, incubation of rat liver microsomes with ATP and Mg2+ decreased the specific activity of HOMeGlt-CoA reductase about tenfold, as determined by immunotitration. The low specific activity produced under these conditions was increased by phosphatase action to nearly the original level. The above evidence suggests that the changes in HOMeGlt-CoA reductase activity that resulted from short-term physiological changes in hormonal or nutritional states of an animal were brought about by a change in the quantity of enzyme, and not by reversible phosphorylation of pre-existing enzyme.

A malonyl-CoA-binding protein from liver.

A soluble protein that binds malonyl-CoA without requiring cofactors has been purified from rat liver. Until saturated, it competes with fatty acid synthetase for free malonyl-CoA, temporarily reducing the rate of fatty acid synthesis at low levels of malonyl-CoA, as in fatty acid synthetase--coupled assays for acetyl-CoA carboxylase. These assays yield low estimates for carboxylase activity with crude and partially purified homogenates containing the malonyl-CoA-binding protein. The protein does not inhibit assays for carboxylase activity that measure nonvolatile radioactivity incorporated from bicarbonate or NADH oxidation coupled to ADP formation. It has an Mr of 180,000 and a subunit of 90,000. It has a lower affinity for ATP, ADP, and acetyl-CoA and none for CO2 or fatty acid synthetase. No enzymatic function has been identified. The protein may regulate malonyl-CoA-binding enzymes.

Kinetic analysis of the individual reductive steps catalyzed by beta-hydroxy-beta-methylglutaryl-coenzyme A reductase obtained from yeast.

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.

Purification of beta-hydroxy-beta-methylglutaryl-coenzyme A reductase from yeast.

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.

Stimulation of hepatic beta-hydroxy-beta-methylglutaryl coenzyme A reductase activity in hypophysectomized rats by L-triiodothyronine.

Diurnally varying activity of hepatic beta-hydroxy-beta-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 mug/100 g of body weight) produced a supranormal level of reductase activity, about 3-4 times the highest activity found in normal rats. Reductase activity began to rise about 30 hr after hormone administration, maintained a constant high level from 48 to 72 hr, and declined to the control level by 96 hr. The supranormal response was elicited equally well either during the day or at night. When normal animals received triiodothyronine, reductase activity was increased only to a level comparable to the highest level found in normal rats. Administration of hydrocortisone markedly inhibited the triiodothyronine-induced increase in reductase activity. This finding suggests that glucocorticoids might act to suppress and thus account for the smaller increase in reductase activity observed in normal rats given triiodothyronine. When actinomycin D was given before the triiodothyronine-induced rise in reductase activity, the increase was effectively blocked, indicating that the hormonal effect requires new RNA synthesis.

High-performance liquid chromatography of coenzyme A esters formed by transesterification of short-chain acylcarnitines: diagnosis of acidemias by urinary analysis.

A protocol for the identification and estimation of short-chain esters of carnitine is described; it is useful for the diagnosis of acidemias. By this method, carnitine esters in urine are converted to coenzyme A esters enzymatically with carnitine acetyltransferase (CAT): short-chain acylcarnitine + CoA cat in equilibrium short-chain acyl-CoA + carnitine. The coenzyme A esters are separated by high-performance liquid chromatography using a radial compression system with a C8 Radial-Pak cartridge and a mobile phase containing 0.025 M tetraethylammonium phosphate in a linear gradient of 1 to 50% methanol. Coenzyme A esters are quantitated by integrator determination of the area under the 254-nm absorption peaks. Enzymatic conversion approaches 100% for acetyl and propionyl esters except in the presence of high levels of free carnitine, which lowers the proportion of ester as acyl-CoA at equilibrium. However, since acidemia patients produce urine low in free carnitine, this problem is minimized. The method is rapid and simple and identifies propionic, methylmalonic, and isovaleric acidemias.