3-hydroxy-3-methylglutaryl-CoA reductase from neonatal chick

The optimal conditions for identification of mevalonic acid as the product of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase are described, as well as the effect of different buffer constituents on the enzyme activity. Under the chosen assay conditions, reductase activity from neonatal chick liver increased with the incubation time up to 60 min and was proportional to the amounts of protein added in a range of 0.1-0.5 mg. The specific activity was maximal in brain and liver and lower in intestine of 6-day-old chicks. Thermostability of hepatic reductase was studied. When microsomal preparations were maintained at 4 degrees C, reductase activity remained unchanged for 6 hr and decreased afterwards. Addition of 50 mM KF to the homogenization medium had no effect on the reductase activity. Similarly, preincubation of microsomal preparations with 105,000 g supernatants in the presence or absence of KF did not significantly increase the reductase activity. These results suggest that HMG-CoA reductase was isolated from neonatal chick in the fully activated form.     

New site-directed reversible inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase.

1. CoA-thioether analogues of 3-hydroxy-3-methylglutaryl-CoA containing an additional methyl group at positions 2, 6(methyl at C3) or 4 of the acyl residue were prepared. To probe for hydrophobic interaction, their inhibitory properties were determined with 3-hydroxy-3-methylglutaryl-CoA reductase purified from baker's yeast. The CoA-thioethers were purely competitive inhibitors whose affinity to the reductase was near to that of the physiological substrate. 2. CoA-sulfoxides derived from the CoA-thioethers displayed affinities to the reductase superior to that of the physiological substrate (Km = 7 microM). Depending on the degree of recognition of diastereomers by the enzyme, the inhibitor constants of the two best inhibitors vary from Ki = 200 nM and Ki = 80 nM (diastereomeric mixtures) to 25 nM and 20 nM, respectively (if only one diastereomer would interact with the enzyme).     

New substrates and inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase

Methyl (RS)-5-bromo-3-hydroxy-3-methyl-pentanoate was prepared by bromination of methyl mevalonate and used for the formation of 4-carboxy-3-hydroxy-3-methylbutyl thioether derivatives by reaction with N-octanoyl-cysteamine, pantetheine, phosphopantetheine and coenzyme A. These thiols were also converted to the (RS)-3-hydroxy-3-methylglutaryl thioester derivatives. The thioesters formed with pantetheine and phosphopantetheine are substrates of 3-hydroxy-3-methylglutaryl-CoA reductase; Km and V values are similar to those of the superior CoA-derivative. The corresponding thioether derivatives in which the oxygen next to sulfur of the substrates is replaced by hydrogen, are inhibitors of the reductase. The inhibition is competitive with 3-hydroxy-3-methylglutaryl-CoA varied, and noncompetitive with NADPH varied. For each of the corresponding pairs of thioester and thioether derivatives Km (substrate) is nearly identical with Ki (inhibitor). The specificity and stereospecificity of the inhibitor action are also shown.     

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA reductase. Characterization and chemical modification

Pseudomonas mevalonii (formerly designated Pseudomonas sp. M (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001; Gill, J. F., Jr., Beach, M.J., and Rodwell, V. W. (1985) J. Biol. Chem. 260, 9393-9398] 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), overexpressed in Escherichia coli (1), has been purified to electrophoretic homogeneity in 75% yield (final specific activity 48 mumols of NAD+ reduced per min/mg protein). The enzyme catalyzes its normal catabolic reaction (mevalonate + 2 NAD+ + CoASH----HMG-CoA + 2NADH + 2H+), and two half-reactions which involve mevaldehyde, the postulated intermediate in the aforementioned reactions and mevaldehyde + NADH + H+----mevalonate + NAD+). The rates of all four reactions and the Michaelis constants for all substrates were measured. Coenzyme A decreased the KM for mevaldehyde reduction 12-fold and stimulated VMAX 2-3 fold. CoASH thus may remain bound throughout the catalytic cycle. Dithiothreitol and analogs of CoASH were tested for their ability to reproduce the CoASH stimulation. Pantetheine, but not dithiothreitol, pantothenate, or desulfo-CoA mimicked CoASH stimulation. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) indicated two sulfhydryl groups per subunit. Both groups remained accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of mevalonate and/or NAD+ but only one group in the presence of HMG-CoA. N-Ethylmaleimide inhibited all the aforementioned reactions. HMG-CoA, but not mevalonate, afforded protection completely and irreversibly inactivated the enzyme. The reactive sulfhydryl group thus may not be a catalytic residue, but may be involved in a conformational change.     

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.     

Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase.

The steady-state level of the resident endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), is regulated, in part, by accelerated degradation in response to excess sterols or mevalonate. Previous studies of a chimeric protein (HM-Gal) composed of the membrane domain of HMGR fused to Escherichia coli beta-galactosidase, as a replacement of the normal HMGR cytosolic domain, have shown that the regulated degradation of this chimeric protein, HM-Gal, is identical to that of HMGR (Chun, K. T., Bar-Nun, S., and Simoni, R. D. (1990) J. Biol. Chem. 265, 22004-22010; Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). Since the cytosolic domain can be replaced with beta-galactosidase without effect on regulated degradation, it has been assumed that the cytosolic domain was not important to this process and also that the membrane domain of HMGR was both necessary and sufficient for regulated degradation. In contrast to our previous results with HM-Gal, we observed in this study that replacement of the cytosolic domain of HMGR with various heterologous proteins can have an effect on the regulated degradation, and the effect correlates with the oligomeric state of the replacement cytosolic protein. Chimeric proteins that are oligomeric in structure are relatively stable, and those that are monomeric are unstable. To test the hypothesis that the oligomeric state of the cytosolic domain of HMGR influences degradation, we use an "inducible" system for altering the oligomeric state of a protein in vivo. Using a chimeric protein that contains the membrane domain of HMGR fused to three copies of FK506-binding protein 12, we were able to induce oligomerization by addition of a "double-headed" FK506-like "dimerizer" drug (AP1510) and to monitor the degradation rate of both the monomeric form and the drug-induced oligomeric form of the protein. We show that this chimeric protein, HM-3FKBP, is unstable in the monomeric state and is stabilized by AP1510-induced oligomerization. We also examined the degradation rate of HMGR as a function of concentrations within the cell. HMGR is a functional dimer; therefore, its oligomeric state and, we predict, its degradation rate should be concentration-dependent. We observed that it is degraded more rapidly at lower concentrations.     

Dietary regulation of 3-hydroxy-3-methylglutaryl-CoA reductase from rat intestine

The effect of dietary cholesterol on rat intestinal 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) varied depending upon whether animals received the dietary cholesterol with polyunsaturated or saturated fats. When cholesterol was fed with polyunsaturates, the enzyme activity in both the jejunum and ileum was significantly suppressed, whereas only the enzyme in the jejunum was significantly suppressed when cholesterol was given with saturated fats. It is concluded that dietary cholesterol has a negative feedback effect on intestinal cholesterol synthesis.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.     

Purification and characterization of fermodulin, an Fe2+-dependent inhibitor protein of 3-hydroxy-3-methylglutaryl-CoA reductase

The activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase of rat liver microsomes was inhibited by the addition of FeSO4 and the cytosolic protein, fermodulin. Modulation of the activity was obtained only in the combined presence of Fe2+ and fermodulin. Using ammonium sulfate fractionation, heat treatment, and chromatography on CM-Sephadex and then on an Fe2+-Blue Sepharose affinity matrix, fermodulin was purified to homogeneity. The molecular weight of the purified protein, as determined by filtration through a Sephacryl S-200 column, was 58,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the protein resolved into two subunits of Mr 43,000 and 28,000. Fermodulin showed ultraviolet absorption and fluorescence spectra typical of tryptophan-containing proteins, and addition of FeSO4 quenched the fluorescence. Using the Millipore filter assay the binding of 1.6 mol 55FeCl2/mol fermodulin was observed in the presence of Tris-HCl buffer. The inhibitory effect of fermodulin at nonsaturating concentrations was potentiated by bicarbonate, ATP.Mg, and ADP.Mg.     

A method for rapid microassay of 3-hydroxy-3-methylglutaryl-CoA reductase activity

A method is devised for the separation of mevalonolactone (MVL) from hydroxymethylglutarate (HMG) in the assay of HMG-CoA reductase activity. The main steps in the procedure consist of absorbing the reaction mixture on the bottom part of a rectangular filter paper and selectively transfering the MVL into the top part of the paper by upward elution with toluene. Under the experimental conditions deseribed, MVL is recovered in an yield of approximately 60%, with little contamination with HMG. Among the advantages of the method are that it involves simple and very few manipulations, no internal standard is required to calculate the recovery of MVL, and simultaneous analyses of a large number of samples are possible.     

Evaluation of 3-hydroxy-3-methylglutaryl-CoA reductase activity by multiple-selected ion monitoring

A new method for the evaluation of 3-hydroxy-3-methylglutaryl-CoA reductase activity is described, based on the multiple-selected ion monitoring of the amount of mevalonate formed in incubations of 3-hydroxy-3-methylglutaryl-CoA with microsomal proteins. Analysis is carried out on crude extracts using deuterated mevalonic acid lactone as internal standard. The sensitivity of the technique allows the quantitative evaluation of mevalonate in microassays (100 μg microsomal protein) of the enzyme activity at the minimum value of the diurnal rhythm.     

The effects of NADPH and 3-hydroxy-3-methylglutaryl-CoA on the thiol/disulfide redox behavior of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase

Microsomal 3-hydroxy-3-methylglutaryl-CoA reductase isolated from the livers of rats fed a diet containing cholestyramine (HMGR-C) is oxidized to a protein-SS-protein disulfide via a thermodynamically favorable thiol/disulfide exchange in glutathione redox buffers which approach the normal in vivo redox poise. In the presence of either substrate (NADPH or 3-hydroxy-3-methylglutaryl-CoA), the equilibrium thiol/disulfide redox behavior of HMGR-C is substantially different than that observed in the absence of substrates or in the presence of both substrates. NADPH present during redox equilibrium in a glutathione redox buffer decreases the equilibrium constant for formation of the protein-SS-protein disulfide (Kox,i) from 0.55 +/- 0.07 M to 0.18 +/- 0.02 M and increases the Kox,m for formation of an inactive protein-SS-glutathione mixed disulfide from less than 1 to 6 +/- 1. The presence of 3-hydroxy-3-methylglutaryl-CoA during redox equilibrium has a similar effect, decreasing the Kox,i for protein-SS-protein disulfide formation to 0.10 +/- 0.02 M and increasing the Kox,m for protein-SS-glutathione mixed disulfide formation to 3.8 +/- 0.9. A three-state model is developed which describes the simultaneous accumulation of protein-SS-protein and protein-SS-glutathione mixed disulfides at redox equilibrium with glutathione redox buffers. Because of the different redox behavior of the free and substrate-liganded forms of the enzyme, addition of 3-hydroxy-3-methylglutaryl-CoA or NADPH to HMGR-C at redox equilibrium results in increased reduction and activation of the enzyme.     

Oxysterol regulators of 3-hydroxy-3-methylglutaryl-CoA reductase in liver. Effect of dietary cholesterol

Hepatic regulatory oxysterols were analyzed to determine which oxysterols were present in livers of mice fed a cholesterol-free diet and whether repression of 3-hydroxy-3-methylglutaryl-CoA reductase following cholesterol feeding was accompanied by an increase in one or more oxysterols. Analysis of free and esterified sterols from mice fed a cholesterol-free diet resulted in the identification and quantitation of six regulatory oxysterols: 24-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, and 7-ketocholesterol. Following the addition of cholesterol to the diet for 1 or 2 nights, hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity declined and the levels of oxysterols, especially those of the side-chain-hydroxylated sterols, increased. Total 3-hydroxy-3-methylglutaryl-CoA reductase repressor units attributable to identified free oxysterols increased 2.5- and 6-fold after 1 and 2 nights, respectively, of cholesterol feeding. The amounts of esterified 24-, 25-, and 26-hydroxycholesterol also increased, with the increase in esterified 24-hydroxycholesterol being the greatest. The 24-hydroxycholesterol was predominantly the 24S epimer and the 26-hydroxycholesterol was predominantly the 25R epimer, indicating enzymatic catalysis of their formation. The observed correlation between increased levels of regulatory oxysterols and repression of 3-hydroxy-3-methylglutaryl-CoA reductase in cholesterol-fed mice is consistent with a hypothesis that intracellular oxysterol metabolites regulate the level of the reductase.     

Rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. Catalysis of the reverse reaction and two half-reactions

Rat liver 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase catalyzes, in addition to its normal biosynthetic or forward reaction (HMG-CoA + 2 NADPH + 2H+----mevalonate + 2 NAD+ + CoASH), the reverse reaction (mevalonate + CoASH + 2 NADP+----HMG-CoA + 2 NADPH + 2H+) and two "half-reactions" that involve the presumed intermediate mevaldate (mevaldate + CoASH + NADP+----HMG-CoA + NADPH + H+ and mevaldate + NADPH + H+----mevalonate + NADP+). These reactions were studied using both enzyme solubilized by the traditional freeze-thaw method and enzyme solubilized with a nonionic detergent in the presence of inhibitors of proteolysis. All four reactions were inhibited by mevinolin, a known inhibitor of the forward (biosynthetic) reaction catalyzed by HMG-CoA reductase. When the enzyme was inactivated by ATP and a cytosolic, ADP-dependent HMG-CoA reductase kinase, the rates of both the forward reaction and the half-reactions decreased to comparable extents. Although coenzyme A is not a stoichiometric participant in the second half-reaction (mevaldate + NADPH + H+----mevalonate + NADP+), it was required as an activator of this reaction. This observation implies that coenzyme A may remain bound to the enzyme throughout the normal catalytic cycle of HMG-CoA reductase.     

Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase.

The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.     

Involvement of calcium in the mevalonate-accelerated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids, is subject to rapid degradation which is regulated by mevalonate (MVA)-derived metabolic products. HMG-CoA reductase is an integral membrane protein of the endoplasmic reticulum, the largest nonmitochondrial pool of cellular Ca2+. To assess the possible role of Ca2+ in the regulated degradation of HMG-CoA reductase, we perturbed cellular Ca2+ concentration and followed the fate of HMG-CoA reductase and of HMGal, a fusion protein consisting of the membrane domain of HMG-CoA reductase and the soluble bacterial enzyme beta-galactosidase. The degradation of HMGal mirrors that of HMG-CoA reductase, demonstrating that the membrane domain of HMG-CoA reductase is sufficient to confer regulated degradation (Skalnik, D.G., Narita, H., Kent, C., and Simoni, R.D. (1988) J. Biol. Chem. 263, 6836-6841; Chun, K.T., Bar-Nun, S., and Simoni, R.D. (1990) J. Biol. Chem. 265, 22004-22010). In this study we show that the MVA-dependent accelerated rates of degradation of HMG-CoA reductase and HMGal in cells maintained in Ca(2+)-free medium are 2-3-fold slower than the rate of degradation in cells grown in high (1.8-2 mM) Ca2+ concentration. This effect is reversed upon addition of Ca2+ to the medium. Furthermore, when cells maintained in high Ca2+ are treated with 1 microM ionomycin, the MVA-dependent accelerated degradation of HMG-CoA reductase and HMGal is also reduced about 2-3-fold. This inhibition is not due to a Ca(2+)-dependent uptake or incorporation of MVA into sterols, since these processes are not affected in the absence of external Ca2+. In addition, cobalt, a known antagonist of Ca(2+)-dependent cellular functions, totally abolishes (IC50 = 520 microM in the presence of 1.8 mM extracellular Ca2+) the MVA-accelerated degradation of HMGal. These results suggest that Ca2+ plays a major role in the regulated degradation of HMG-CoA reductase.