Inhibition of mevalonate 5-diphosphate decarboxylase by fluorinated substrate analogs

Mevalonate 5-diphosphate decarboxylase (MDD) is a peroxisomal enzyme in the cholesterol biosynthetic pathway, which plays an important role in regulating cholesterol biosynthesis. In the present study, rat MDD was cloned and purified to apparent homogeneity. Two fluorinated MDD substrate analogs, P'-geranyl 2-fluoromevalonate 5-diphosphate (4) and 2-fluoromevalonate 5-diphosphate (6), were synthesized, and both were found to be irreversible inhibitors of rat MDD. These two inhibitors were characterized, and mechanisms of the inactivation process were proposed. Kinetic studies indicate both analogs only bind into mevalonate binding-site of MDD. Compound 4 shows competitive inhibition on mevalonate kinase (MVK), and its IC(50) value was determined to be comparable with that of geranyl diphosphate. Further kinetic studies indicate compound 4 only bind into ATP binding-site of MVK. These studies provide an example for a single inhibitor to carry out sequential blocking of two enzymes in cholesterol biosynthesis, which may provide useful information for drug discovery for the purpose of treating cardiovascular disease and cancer or for pest control.     

Substrate binding order in mevalonate 5-diphosphate decarboxylase from chicken liver

The substrate binding order of chicken liver mevalonate 5-diphosphate decarboxylase was investigated by using competitive inhibitors of the substrates. Mevalonate and mevalonate 5-phosphate showed mixed inhibition when ATP was the varied substrate. Both analogues of ATP, adenosine 5'-O-(3-thiotriphosphate) and beta-tau methylene adenosine 5'-triphosphate showed uncompetitive inhibition against mevalonate 5-diphosphate. These results are in accordance with an ordered sequential mechanism with mevalonate 5-diphosphate as the first substrate to bind to the enzyme.     

Mevalonate-5-diphosphate decarboxylase: stereochemical course of ATP-dependent phosphorylation of mevalonate 5-diphosphate

Chicken liver mevalonate-5-diphosphate decarboxylase catalyzes the reaction of mevalonate 5-diphosphate (MVADP) with ATP to produce isopentenyl diphosphate, ADP, CO2, and inorganic phosphate. The overall reaction involves an anti elimination of the tertiary hydroxyl and carboxyl groups. To investigate the mechanism for transfer of the terminal phosphoryl group of ATP to the C-3 oxygen of MVADP, we have carried out the reaction using stereospecifically labeled (Sp)-adenosine 5'-O-(3-thio[3-17O2,18O]triphosphate) [( gamma-17O2,18O]ATP gamma S) in place of ATP. The configuration of the [17O,18O]thiophosphate produced was found to be Rp, corresponding to overall inversion of configuration at phosphorus in the thiophosphoryl group transfer step. This result is consistent with the direct transfer of the thiophosphoryl group from (Sp)-[gamma-17O2,18O]ATP gamma S to MVADP at the active site. Our result does not indicate the involvement of a covalent thiophosphoryl-enzyme on the reaction pathway.     

Mevalonate kinase is localized in rat liver peroxisomes.

Recent data suggest that rat liver peroxisomes play a critical role in cholesterol synthesis. Specifically, peroxisomes contain a number of enzymes required for cholesterol synthesis as well as sterol carrier protein-2. Furthermore, peroxisomes are involved in the in vitro synthesis of cholesterol from mevalonate and contain significant levels of apolipoprotein E, a major constituent of several classes of plasma lipoproteins. In this study we have investigated the subcellular localization of mevalonate kinase (EC 2.7.1.36; ATP:mevalonate-5-phosphotransferase). Mevalonate kinase is believed to be a cytosolic enzyme and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. Mevalonate kinase has been purified from rat liver cytosol and a cDNA clone coding for rat mevalonate kinase has also been isolated and characterized. In this study, utilizing monoclonal antibodies made against the purified rat mevalonate kinase, we demonstrate the presence of mevalonate kinase in rat liver peroxisomes and in the cytosol. Each of these compartments contained a different form of the protein. The pI and the Mr of the peroxisomal protein is 6.2 and 42,000, respectively. The pI and Mr of the cytosolic protein is 6.9 and 40,000, respectively. The peroxisomal protein was also significantly induced by a number of different hypolipidemic drugs. In addition, we present evidence for the unexpected finding that the purified mevalonate kinase (isolated from the cytosol and assumed to be a cytosolic protein) is actually a peroxisomal protein.     

Purification and regulation of mevalonate kinase from rat liver

Mevalonate kinase may play a key role in regulating cholesterol biosynthesis because its activity may be regulated via feedback inhibition by intermediates in the cholesterol biosynthetic pathway. To study the regulation of mevalonate kinase, the enzyme was purified to homogeneity from rat liver, and monospecific antibody against mevalonate kinase was prepared. The purified mevalonate kinase had a dimeric structure composed of identical subunits, and the Mr of the enzyme determined by gel chromatography was 86,000. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit Mr was 39,900. The pI for mevalonate kinate was 6.2. The levels of mevalonate kinase protein and enzyme activity were determined in the livers of rats treated with either cholesterol-lowering agents (cholestyramine, pravastatin, and lovastatin) or with dietary modifications. Diets containing cholestyramine alone or cholestyramine and either pravastatin or lovastatin increased mevalonate kinase activity 3-6-fold. Mevalonate kinase activity decreased approximately 50% in rats treated with diets containing either 5% cholesterol or 5% cholesterol and 0.5% cholic acid. Fasting did not significantly change mevalonate kinase activity. The amount of mevalonate kinase protein in the liver was quantitated using immunoblots, and the changes in the levels of kinase activity induced by either drug treatment or by cholesterol feeding were correlated with similar changes in the levels of mevalonate kinase protein. Therefore, under these experimental conditions, mevalonate kinase activity in the liver was regulated principally by changes in the rates of enzyme synthesis and degradation.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse uterine epithelial cells.

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase was studied in mouse uterine epithelium. The enzyme was rapidly inactivated during incubation with ATP/Mg2+ in vitro, and could be re-activated by incubation with partially purified rat liver phosphoprotein phosphatase. Enzyme activity was rapidly inhibited by mevalonate injection in vivo to approx. 30% of control. The percentage of total enzyme active in vivo was measured by inclusion of NaF in the isolation buffers. The percentage of enzyme active in vivo 18 h after stimulation by oestrogens remained at approx. 25% after inhibition of activity by mevalonate injection, cholesterol feeding or progesterone pretreatment. However, 9 h after oestrogen stimulation, cholesterol feeding inhibited enzyme activity to 57% of control, 94% of which was in the active form. We conclude that, although all components for a reversible phosphorylative regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity are present in uterine epithelial cells, a role in the rapid changes in epithelial enzyme activity has not been demonstrated.     

Improved procedures for the synthesis of phosphomevalonate and for the assay and purification of pig liver phosphomevalonate kinase

An improved procedure for the synthesis of phosphomevalonate using excess free ATP4-, and phenyl agarose to remove contaminating nucleotides, is described. A high-voltage electrophoresis assay, which separates phosphomevalonate from mevalonate 5-diphosphate at pH 3.5, was developed for the assay of phosphomevalonate kinase (ATP:5-phosphomevalonate phosphotransferase, EC 2.7.4.2). High-voltage electrophoresis, at pH 5, could also be used for the separation of mevalonate 5-diphosphate from isopentenyl diphosphate. An alternative method for the purification of phosphomevalonate kinase from pig liver was also developed. The high-voltage electrophoresis assay was used to reassess the metal ion and nucleotide specificity of the pig liver phosphomevalonate kinase. ATP could be partially replaced by ITP and GTP and poorly by CTP and UTP. Apparent activation of the enzyme by free ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224).     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

The role of circulating mevalonate in nephrotic hypercholesterolemia in the rat

The cause of the hypercholesterolemia that characterizes the nephrotic syndrome has never been adequately explained. The present study examines the possibility that enhanced availability of the cholesterol precursor, mevalonic acid, to the liver in the nephrotic state may result in increased hepatic cholesterogenesis. In normal animals, the kidneys are known to be the major site of the metabolism of circulating mevalonate to both cholesterol and CO2. Previous studies, using perfusion of isolated, intact kidneys, have shown that the excretion and metabolism of mevalonate are both impaired in nephrosis. The present investigation has demonstrated in vivo that puromycin aminonucleoside nephrosis results in a 25% reduction in the oxidation of mevalonate to CO2. In the same nephrotic animals, cholesterogenesis from circulating mevalonate was significantly increased in both liver and carcass. In addition, liver slices from nephrotic animals incorporated increased amounts of [5-14C]mevalonate into cholesterol when calculated per whole liver, but not per gram of liver. Oxidation of mevalonic acid by kidney slices was significantly reduced, whether expressed as per gram of tissue or per whole organ. HMG-CoA (3-hydroxy-3-methylglutaryl) reductase activity in liver of nephrotic animals was significantly increased. We conclude that, in the nephrotic state, impaired mevalonate metabolism by the kidney may contribute to enhanced cholesterogenesis by increasing delivery of mevalonate to liver and carcass; in addition, nephrosis appears to provide an undefined stimulus for HMG-CoA reductase activity in the liver, thereby providing an additional enhancement of hepatic cholesterogenesis.     

Hypolipidemic activity of dipyridamole: effects on the main regulatory enzyme of cholesterogenesis.

The in vivo dipyridamole treatment for 16 days produced a significant decrease in chick plasma cholesterol, mainly due to the esterified form. This effect was especially patent in the VLDL + LDL fraction. Similar results were observed in triglyceride content. To our knowledge, this is the first report on this hypolipidemic effects of dipyridamole. Total and esterified cholesterol increased after the same treatment in chick liver, while brain cholesterol content was not affected. Hepatic 3-hydroxy-3- methylglutaryl-CoA reductase activity was drastically reduced, while other secondary regulatory enzymes such as mevalonate kinase, mevalonate 5-phosphate kinase and mevalonate 5-pyrophosphate decarboxylase did not change significantly. No significant differences were found in cholesterol and lipidic phosphorus from liver microsomes, so that the effect of dipyridamole on reductase activity cannot be due to modifications in cholesterol/lipidic phosphorus molar ratio. Neither of these enzyme activities was affected in vitro by dipyridamole.     

A conserved cysteine in molybdenum oxotransferases

The amino acid sequences of peptides derived from rat hepatic sulfite oxidase have been determined by a combination of amino acid analysis and Edman degradation of the purified protein. The data obtained showed the rat liver enzyme contained 3 cysteine residues which was confirmed by thiol modification studies using 4,4'-dithiodipyridine of the native enzyme. Combining these data with that previously published for chicken liver sulfite oxidase (Neame, P. J., and Barber, M. J. (1989) J. Biol. Chem. 264, 20894-20901) indicates that 2 cysteines (Cys186 and Cys430, based upon the numbering for the chicken sequence) are conserved in both chicken and rat liver enzymes with all the cysteine residues being present in the molybdenum-containing domain. Further comparison of the sequences of the molybdenum domains of rat and chicken liver sulfite oxidase with the amino acid sequences published for the molybdenum domains of a variety of assimilatory nitrate reductases suggests that only a single cysteine residue (Cys186) is conserved in all these enzymes, indicating that it may play a role in the binding of Mo-pterin to the protein.     

Kinetic characterization of an oxidative,cooperative HMG-CoA reductase from Burkholderia cenocepacia

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different V_max values.     

The isolation of 2,3-oxidosqualene from the liver of rats treated with 1-dodecylimidazole, a novel hypocholesterolaemic agent

1. Non-saponifiable lipid from the livers of rats treated with 1-dodecylimidazole contained an unidentified compound that was not present in the livers from untreated animals. 2. Treated rats had lower serum cholesterol concentrations than control rats. 3. 1-Dodecylimidazole, when added to rat liver slices, inhibited the incorporation of [1-(14)C]acetate and [2-(14)C]mevalonate into digitonin-precipitable sterols and resulted in the accumulation of a labelled compound, which was chromatographically identical with the unknown compound described in 1 above. 4. Rats treated with 1-dodecylimidazole incorporated less [(14)C]mevalonate into liver digitonin-precipitable sterols than untreated animals and accumulated the unknown compound as a labelled intermediate. 5. The unknown intermediate had the same chromatographic properties, n.m.r. and mass spectra as authentic 2,3-oxidosqualene. 6. The identity of the intermediate as 2,3-oxidosqualene was further established by showing that it was incorporated into sterols by rat liver homogenates under anaerobic conditions. In addition, incubation of [(14)C]squalene with rat liver homogenates resulted in trapping of the radioactivity by the added intermediate. 7. It is suggested that the hypocholesterolaemic activity of 1-dodecylimidazole results in part from the inhibition of cholesterol biosynthesis at the level of 2,3-oxidosqualene sterol cyclase.     

Effects of peroxisome proliferators gemfibrozil and clofibrate on syntheses of dolichol and cholesterol in rat liver

The effects of two peroxisome proliferators, gemfibrozil and clofibrate, on syntheses of dolichol and cholesterol in rat liver were investigated. Gemfibrozil did not affect the overall content of dolichyl phosphate, but it changed the chain-length distribution of dolichyl phosphate, increasing the levels of species with shorter isoprene units. Gemfibrozil suppressed synthesis of dolichyl phosphate from [(3)H]mevalonate and [(3)H]farnesyl pyrophosphate in rat liver. In contrast, clofibrate increased the content of dolichol (free and acyl ester forms). It remarkably enhanced dolichol synthesis from mevalonate, but did not affect dolichol synthesis from farnesyl pyrophosphate. Gemfibrozil elevated cholesterol synthesis from [(14)C]acetate, but did not affect the synthesis from mevalonate. Clofibrate suppressed cholesterol synthesis from acetate, but did not affect cholesterol synthesis from mevalonate. These results suggest that gemfibrozil suppresses synthesis of dolichyl phosphate by inhibiting, at the least, the pathway from farnesyl pyrophosphate to dolichyl phosphate. As a result, the chain-length pattern of dolichyl phosphate may show an increase in shorter isoprene units. Clofibrate may increase the content of dolichol by enhancing dolichol synthesis from mevalonate. Gemfibrozil may increase cholesterol synthesis by activating the pathway from acetate to mevalonate. Unlike gemfibrozil, clofibrate may decrease cholesterol synthesis by inhibiting the pathway from acetate to mevalonate.     

Characterization of phosphomevalonate kinase: chromosomal localization, regulation, and subcellular targeting

Phosphomevalonate kinase catalyzes the conversion of mevalonate-5-phosphate to mevalonate-5-diphosphate and was originally believed to be a cytosolic enzyme. In this study we have localized the phosphomevalonate kinase gene to chromosome 1p13-1q22-23 and present a genomic map indicating that the gene spans more than 8.4 kb in the human genome. Furthermore, we show that message levels and enzyme activity of rat liver phosphomevalonate kinase are regulated in response to dietary sterol levels and that this regulation is coordinate with 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol biosynthesis. In addition, we demonstrate that phosphomevalonate kinase is a peroxisomal protein which requires the C-terminal peroxisomal targeting signal, Ser-Arg-Leu, for localization to the organelle.     

Inactivation of chicken liver mevalonate 5-diphosphate decar☐ylase by sulfhydryl-directed reagents: evidence of a functional dithiol

Chicken liver mevalonate 5-diphosphate decar☐ylase (ATP:(R)-5-diphosphomevalonate car☐y-lyase (dehydrating), EC 4.1.1.33) is inactivated by methylmethanethiosulfonate and 5,5′-dithiobis(2-nitrobenzoate). The presence of the substrates ATP or mevalonate 5-diphosphate protect very effectively against inactivation. The inactivation is second order with respect to methylmethanethiosulfonate, with an inactivation rate constant of (7.6 ± 0.1) · 10⁻⁵ μM⁻² ·s⁻¹, implying that the modifier may be reacting with more than one thiol in the enzyme. The enzyme is also inactivated by a number of dithiol-specific reagents, suggesting the presence of a functional dithiol. The determined pK_app values for enzyme modification by methyl methanethiosulfonate and phenylarsine oxide are 7.3 ± 0.1 and 7.6 ± 0.3, respectively. From the data presented, it is concluded that the enzyme posseses a functional dithiol that is important for substrate binding.     

Selective inhibition of cholesterol synthesis by cell-free preparations of rat liver by using inhibitors of cytoplasmic acetoacetyl-coenzyme A thiolase.

Cytoplasmic acetoacetyl-CoA thiolase (acetyl-CoA C-acetyltransferase, EC 2.3.1.9) was partially purified from rat liver. The enzyme was irreversibly inactivated by 4-bromocrotonyl-CoA, but-3-ynoyl-CoA, pent-3-ynoyl-CoA and dec-3-ynoyl-CoA. In the case of the alk-3-ynoyl-CoA esters the potency as alkylating agents of acetoacetyl-CoA thiolase decreased with increased chain length of the alk-3-ynoyl moiety. Advantage was taken of the specific action of alk-3-ynoyl-CoA esters on acetoacetyl-CoA thiolase to show that in a postmitochondrial fraction from rat liver they are effective inhibitors of cholesterol synthesis from sodium [2-14C]acetate under conditions when mevalonate conversion into cholesterol and fatty acid synthesis are unafffected. Short-chain alk-3-ynoic acids have little effect on sterol synthesis, although dec-3-ynoic acid is an effective inhibitor owing to its conversion into the CoA ester by the microsomal fatty acyl-CoA synthetase.     

Effect of a disulfide bond on mevalonate kinase

Mevalonate kinase is one of ATP-dependent enzymes in the mevalonate pathway and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. In animal cells, it plays a key role in regulating biosynthesis of cholesterol, while in microorganisms and plants, it is involved in the biosynthesis of isoprenoid derivatives that are one of the largest groups of natural products. Crystal structure and sequence alignment show that a unique disulfide bond exists in mevalonate kinase of thermostable species Methanococcus jannaschii, but not in rat mevalonate kinase. In the present study, we investigated the effect of the disulfide bond in M. jannaschii mevalonate kinase and an engineered disulfide bond in rat mevalonate kinase mutant A141C on the properties of enzymes through characterization of their wild-type and variant enzymes. Our result suggests that the Cys107-Cys281 disulfide bond is important for maintaining the conformation and the thermal activity of M. jannaschii mevalonate kinase. Other interactions could also have contributions. The thiol-titration and fluorescence experiment further indicate that rat mevalonate kinase A141C variant enzyme has a new disulfide bond, which makes the variant protein enhance its thermal activity and resist to urea denaturation.     

Mutation and inhibition studies of mevalonate 5-diphosphate decarboxylase

Mevalonate 5-diphosphate decarboxylase plays an important role in regulating cholesterol biosynthesis, which was studied through incubation with various synthetic substrate analogs and characterization of mutated enzymes. The results are potentially useful for further developing inhibitors that block the mevalonate pathway which is a drug target for treating cardiovascular disease and cancer.