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1.
K M Gibson G Hoffmann A Schwall R L Broock S Aramaki L Sweetman W L Nyhan I K Brandt R S Wappner W Lehnert 《Journal of lipid research》1990,31(3):515-521
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was measured in extracts of cultured fibroblasts derived from patients with mevalonate kinase deficiency (MKD). For six patients studied, the mean activity of 63.3 +/- 41.1 pmol/min-mg protein (+/- 1 SD, range 37.7-146.2) was significantly higher than the mean value in three control fibroblast lines of 11.1 +/- 3.5 (+/- 1 SD, range 8.0-14.9). These values were obtained using cells subcultured in medium supplemented with 10% fetal bovine serum (FBS) 21 h prior to assay. When cells were deprived of cholesterol by subculturing for 21 h in delipidated FBS, the mean value for patient cells was increased to 230.8 +/- 78.5 pmol/min-mg protein (range 130.9-333.8) as compared to 109.5 +/- 47.1 (range 78.0-163.6) for controls. The activity of HMG-CoA synthase in extracts of fibroblasts derived from the patients was not elevated. The mevalonic acid concentration in the surrounding culture medium was assessed by stable isotope dilution assay. For five patients, the mean concentration in medium containing FBS was 0.92 +/- 0.37 microM (+/- 1 SD, range 0.46-1.48) in contrast to 1.24 +/- 0.83 microM (range 0.46-2.54) for cells subcultured in delipidated FBS. The mean value for three control fibroblast lines was 0.22 +/- 0.12 microM (+/- 1 SD, range 0.11-0.35) for cells subcultured in FBS as compared to 0.01 +/- 0.01 microM (range 0.0-0.01 microM) for cells sucultured in delipidated FBS.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5 alpha-reductase from female rats was determined to be equilibrium ordered sequential by initial velocity, product inhibition and dead-end inhibition studies. Analysis of the initial velocity data resulted in intersecting double reciprocal plots indicating a sequential mechanism (apparent Km (progesterone) = 95.4 +/- 4.5 nM; apparent Kia(NADPH) = 9.9 +/- 0.7 microM). The plot of 1/v vs 1/progesterone intersected on the ordinate which is consistent with an equilibrium ordered mechanism. Ordered addition of the substrates was also supported by product inhibition studies with NADP versus NADPH and NADP versus progesterone. NADP is a competitive inhibitor versus NADPH (apparent Kis = 4.3 +/- 1.3 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 31.9 +/- 1.4 microM and apparent Kii = 145.4 +/- 15.5 microM). These inhibition patterns show that NADPH binds prior to progesterone. Taken together, these analyses indicate that the cofactor, NADPH, binds to the enzyme in rapid equilibrium and preferentially precedes the binding of progesterone. 相似文献
3.
Altered kinetic properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase following dietary manipulations 总被引:4,自引:0,他引:4
The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the rate-limiting step in the cholesterogenic pathway and was proposed to be composed in situ of 2 noncovalently linked subunits (Edwards, P.A., Kempner, E.S., Lan, S.-F., and Erickson, S.K. (1985) J. Biol. Chem. 260, 10278-10282). In the present report, the activities and kinetic properties of HMG-CoA reductase in microsomes isolated from livers of rats fed on diets supplemented with either ground Amberlite XAD-2 ("X"), cholestyramine/mevinolin ("CM"), or unsupplemented, normal rat chow ("N"), were compared. The specific activities of HMG-CoA reductase in X and CM microsomes were, respectively, 5- and 83-fold higher than that of N microsomes. In NADPH-dependent kinetics of HMG-CoA reductase activated with 4.5 mM GSH, the concentration of NADPH required for half-maximal velocity (S0.5) was 209 +/- 23, 76 +/- 23, and 40 +/- 4 microM for the N, X, and CM microsomes, respectively. While reductase from X microsomes displays cooperative kinetics toward NADPH (Hill coefficient (nH) = 1.97 +/- 0.07), the enzyme from CM microsomes does not (nH = 1.04 +/- 0.07). Similarly to HMG-CoA reductase from CM microsomes, the freeze-thaw solubilized enzyme ("SOL") displays no cooperativity toward NADPH and its Km for this substrate is 34 microM. At 4.5 mM GSH, HMG-CoA reductase from X, CM, and SOL preparations has a similar Km value for [DL]-HMG-CoA, ranging between 13-16 microM, while reductase from N microsomes had a higher Km value (42 microM) for this substrate. No cooperativity towards HMG-CoA was observed in any of the tested enzyme preparations. Immunoblotting analyses of the different preparations demonstrated that the observed altered kinetics of HMG-CoA reductase in the microsomes is not due to preferential proteolytic cleavage of the native 97-100 kDa subunit of the enzyme to the noncooperative 50-55 kDa species. Moreover, it was found that the ratio enzymatic activity/immunoreactivity of the reductase increased in the order N less than X less than CM approximately equal to SOL, indicating that the activity per reductase molecule increases with the induction of the enzyme. These results are compatible with a model suggesting that dietary induction of hepatic HMG-CoA reductase may change the state of functional aggregation of its subunits. 相似文献
4.
An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone. 相似文献
5.
1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-carboxylic acid (QCA), 1-isoquinolinecarboxylic acid (IQCA), 2,2'-bi-1H-imidazole (2,2'-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine beta-hydroxylase with respect to ascorbate (Kis = 5.7(+/- 0.9) microM, 0.14(+/- 0.03) mM, 0.80(+/- 0.20) mM; nH = 1.4(+/- 0.1), 1.8(+/- 0.4), 2.8(+/- 0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine beta-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine beta-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (Kis = 1.9(+/- 0.3) microM, 21(+/- 6) microM, and 0.9(+/- 0.3) microM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine beta-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine beta-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine beta-hydroxylase. 相似文献
6.
Mode of interaction of beta-hydroxy-beta-methylglutaryl coenzyme A reductase with strong binding inhibitors: compactin and related compounds 总被引:2,自引:0,他引:2
The sodium salts of compactin (1) and trans-6-[2-(2,4- dichloro-6-hydroxyphenyl)ethyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran- 2-one (3) are inhibitors of yeast beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase. The dissociation constants are 0.24 X 10(-9) and 0.28 X 10(-9) M, respectively. Similar values have been reported for HMG-CoA reductase from mammalian sources [Endo, A., Kuroda, M., & Tanzawa, K. (1976) FEBS Lett. 72, 323; Alberts, A. W., et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3957]. The structures of these compounds marginally resemble that of any substrates of HMG-CoA reductase. We, therefore, investigated the basis for the strong interaction between HMG-CoA reductase and these inhibitors. HMG-CoA and coenzyme A (CoASH), but not reduced nicotinamide adenine dinucleotide phosphate (NADPH), prevent binding of compactin to the enzyme. HMG-CoA, but not CoASH or NADPH, prevents binding of 3 to the enzyme. We also investigated the inhibitory activity of molecules that resemble structural components of compactin. Compactin consists of a moiety resembling 3,5-dihydroxyvaleric acid that is attached to a decalin structure. The sodium salt of DL-3,5-dihydroxyvaleric acid inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The dissociation constant for DL-3,5-dihydroxyvaleric acid, derived from protection against inactivation of enzyme by iodoacetic acid, is (2.1 +/- 0.9) X 10(-2) M. Two decalin derivatives (structurally identical with or closely related to the decalin moiety of compactin) showed no detectable inhibition. If the lack of inhibition is due to their limited solubility, the dissociation constant of these decalin derivatives may be conservatively estimated to be greater than or equal to 0.5 mM. Simultaneous addition of decalin derivatives and DL-3,5-dihydroxyvaleric acid does not lead to enhanced inhibition. The sodium salt of (E)-6-[2-(2-methoxy-1-naphthalenyl)ethenyl]-3,4,5,6- tetrahydro-4-hydroxy-2H-pyran-2-one (6) inhibits HMG-CoA reductase competitively with respect to HMG-CoA and noncompetitively with respect to NADPH. The inhibition constant (vs. HMG-CoA) is 0.8 microM. CoASH does not prevent binding of 6 to enzyme. Compound 6, therefore, behaves analogously to compound 3. We propose that these inhibitors occupy two sites on the enzyme: one site is the hydroxymethylglutaryl binding domain of the enzyme active site and the other site is a hydrophobic pocket located adjacent to the active site.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
7.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man. 相似文献
8.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli. 总被引:5,自引:1,他引:4
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Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism. 相似文献
9.
Involvement of calcium in the mevalonate-accelerated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase 总被引:4,自引:0,他引:4
J Roitelman S Bar-Nun S Inoue R D Simoni 《The Journal of biological chemistry》1991,266(24):16085-16091
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. 相似文献
10.
T N Pajewski F D Pinkerton L R Miller G J Schroepfer 《Chemistry and physics of lipids》1988,48(3-4):153-168
The metabolism of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I) has been studied in Chinese hamster ovary (CHO-K1) cells which were maintained in a lipid-deficient medium. The incorporation of I into the cells was linear with respect to sterol concentration in the medium over the ranges of concentrations studied and was more than 3.5 times that of the uptake of cholesterol. The results of detailed chromatographic analyses of the lipids recovered from the cells after 6 h of incubation with [2,4-3H]I (0.5 microM or 6.0 microM) indicated that most of the 3H was associated with free I. Considerably lesser amounts of the 3H was associated with esters of I. No formation of [3H]cholesterol or [3H]cholesteryl esters (or other C27 monohydroxysterols) from labeled I was observed. The labeled material with the chromatographic behavior of the esters of I gave, after mild alkaline hydrolysis, the free 15-ketosterol which was characterized by the results of chromatographic and cocrystallization studies. Upon transfer of the CHO-K1 cells from a culture medium containing 8% newborn calf serum to the same medium containing 8% lipid-deficient newborn calf serum, increases in the levels of activity of cytosolic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and of HMG-CoA reductase were observed. These increases were blocked by the addition of I at a concentration of 1.0 microM. I (1.0 microM) also caused a decrease in the levels of activity of the three enzymes in cells previously grown in medium containing lipid-deficient serum. These results demonstrate that I not only affects the enzymatic reduction of HMG-CoA but also the enzymatic formation of this key intermediate in cholesterol biosynthesis. 相似文献
11.
Serfass L Carr HS Aschenbrenner LM Burstyn JN 《Archives of biochemistry and biophysics》2001,387(1):47-56
The inhibition of soluble guanylyl cyclase by Ca2+ has been kinetically characterized and the results support a two-metal-ion catalytic mechanism for formation of cGMP. Ca2+ reversibly inhibits both the basal and NO-stimulated forms of bovine lung soluble guanylyl cyclase. Inhibition is independent of the activator identity and concentration, revealing that Ca2+ interacts with a site independent of the heme regulatory site. Inhibition by Ca2+ is competitive with respect to Mg2+ in excess of substrate, with Kis values of 29 +/- 4 and 6.6 +/- 0.6 microM for the basal and activated states, respectively. Ca2+ inhibits noncompetitively with respect to the substrate MgGTP in both activity states. The qualitatively similar inhibition pattern and quantitatively different Ki values between the basal and NO-stimulated states suggest that the Ca2+ binding site undergoes some structural modification upon activation of the enzyme. The competitive nature of Ca2+ inhibition with respect to excess Mg2+ is consistent with a two-metal-ion mechanism for cyclization. 相似文献
12.
Rabbits were given 50 i.u. hCG, i.v., to initiate ovulation and pseudopregnancy (Day 0) and were treated, s.c., with or without a 1-cm Silastic oestradiol implant. Serum progesterone concentrations were measured at 4-day intervals and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity was estimated by the conversion of HMG to mevalonate in microsomes from corpora lutea removed on Days 4, 8, 12, 16 and 20 of pseudopregnancy (4 rabbits/day). Total HMG-CoA reductase activity was significantly (P less than 0.05) higher in control rabbits on Days 8 and 12 (5.29 +/- 0.63 and 5.5 +/- 0.28 nmol/min/mg protein, respectively) compared to oestradiol-treated rabbits (2.57 +/- 0.25 and 4.03 +/- 0.23 nmol/min/mg protein, respectively). On Days 16 and 20, total HMG-CoA reductase activity was not different in control and oestradiol-treated animals. There was no difference in the levels of the active fraction of HMG-CoA reductase, which represented less than 20% of the total enzyme activity, in control and oestradiol-treated rabbits (less than 780 pmol/min/mg protein, Day 12). These results indicate that oestradiol does not alter the active form, but can reduce the total activity of HMG-CoA reductase in the rabbit corpus luteum without a decline in serum progesterone. Therefore, neither total nor active forms of HMG-CoA reductase are directly related to progesterone secretion. This suggests that other sources of cholesterol may contribute to progesterone production in the rabbit. 相似文献
13.
Ohashi K Osuga J Tozawa R Kitamine T Yagyu H Sekiya M Tomita S Okazaki H Tamura Y Yahagi N Iizuka Y Harada K Gotoda T Shimano H Yamada N Ishibashi S 《The Journal of biological chemistry》2003,278(44):42936-42941
The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/-) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/- embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/- mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/- and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis. 相似文献
14.
Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase,an enzyme of isopentenyl diphosphate biosynthesis
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Sutherlin A Hedl M Sanchez-Neri B Burgner JW Stauffacher CV Rodwell VW 《Journal of bacteriology》2002,184(15):4065-4070
Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis. 相似文献
15.
Tomoda H Ohbayashi N Kumagai H Hashizume H Sunazuka T Omura S 《Biochemical and biophysical research communications》1999,265(2):536-540
A synthetic beta-lactone trans-DU-6622 (3-hydroxy-2-(hydroxymethyl)-5-[7-(methylcarbonyl)-naphthalen++ +-1-yl]pentanoic acid 1,3-lactone, a mixture of (2R, 3R)- and (2S, 3S)-beta-lactones) was found to inhibit HMG-CoA synthase (IC(50): 0. 15 microM) and pancreatic lipase (IC(50): 120 microM). The effects of the optically pure DU-6622 isomers on the two enzymes were compared. The (2R, 3R)-isomer was shown to be a highly specific inhibitor of HMG-CoA synthase (IC(50): 0.098 microM vs 270 microM for pancreatic lipase), while the (2S, 3S)-isomer markedly increased the specificity of lipase inhibition (IC(50): 27 microM vs 31 microM for HMG-CoA synthase). Furthermore, the (2R, 3R)-isomer strongly inhibited the binding of [(14)C]hymeglusin to HMG-CoA synthase, indicating that the isomer was bound to the same site of the synthase as hymeglusin. The (2R, 3R)-beta-lactone is responsible for the specific inhibition of HMG-CoA synthase, while the (2S, 3S)-beta-lactone is responsible for the inhibition of pancreatic lipase. 相似文献
16.
Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol. 相似文献
17.
Isoflavones identified as inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in soybean paste were assayed using the catalytic portion of Syrian hamster HMG-CoA reductase, and the kinetic values were measured using HMG-CoA and NADPH. The inhibition of HMG-CoA reductase by these inhibitors was competitive with HMG-CoA and noncompetitive with NADPH. Ki values for genistein, daidzein, and glycitein were 27.7, 49.5, and 94.7 microM, respectively. 相似文献
18.
3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product. 总被引:1,自引:0,他引:1
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D A Bochar J R Brown W F Doolittle H P Klenk W Lam M E Schenk C V Stauffacher V W Rodwell 《Journal of bacteriology》1997,179(11):3632-3638
The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C. 相似文献
19.
3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue. 相似文献
20.
Adenosine 2'-monophospho-5'-diphosphoribose (P-ADP-Rib) is a structural analog of NADPH which was reported to competitively inhibit (Kiapp = 21.7 microM) solubilized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Tanazawa, K., and A. Endo. 1979. Eur. J. Biochem. 98: 195-201). However, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib (Kiapp = 550 microM at 4.5 mM GSH). Gradual shift of the microsomal reductase towards a non-allosteric form by increasing glutathione (GSH) concentrations resulted in a higher inhibition by P-ADP-Rib. Under these conditions, Ki values for P-ADP-Rib were 165 microM and 53 microM at 9 mM and 27 mM GSH, respectively. The largest change in the degree of inhibition by P-ADP-Rib was observed within the 10 mM range of GSH. By contrast, freeze-thaw solubilized HMG-CoA reductase, which does not display allosteric properties, is readily inhibited by P-ADP-Rib, even when assayed at a low concentration of GSH (Kiapp = 50 microM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP-Rib (22 microM at 27 mM GSH). Microsomal HMG-CoA reductase is allosterically activated by various nucleotides. When activated by NADH, the enzyme is effectively inhibited by P-ADP-Rib even at a 4.5-mM GSH concentration (Kiapp = 175 microM in the presence of 300 microM NADH).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献