Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.     

3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Purification, molecular and catalytic properties.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified to homogeneity from ox liver and obtained essentially free from acetoacetyl-CoA thiolase activity. The purification procedure included substrate elution from cellulose phosphate and chromatofocusing. The relative molecular mas was about 100 000 and S20,w0 was 6.36S. The enzyme appears to be a dimer of identical subunits (Mr 47 900). The Km for acetoacetyl-CoA is extremely low (less than 0.5 microM), and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition (KiAcac-CoA = 3.5 microM) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-hydroxy-3-methylglutaryl-CoA give mixed product inhibition with respect to acetyl-CoA, which is compatible with a Ping Pong mechanism in which both products can form kinetically significant complexes with two forms of the enzyme. The two forms are most likely to be free enzyme and an acetyl-enzyme intermediate.     

Enzymes involved in acetoacetate formation in various bovine tissues

1. The activities of acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase and lyase and acetoacetyl-CoA deacylase were measured in homogenates of samples of liver, rumen epithelium (long papillae), kidney and lactating mammary gland derived from slaughtered cows. 2. The activities of the four enzymes in bovine liver were similar to the activities previously reported for the corresponding enzymes in rat liver. 3. Acetoacetyl-CoA thiolase and hydroxymethylglutaryl-CoA synthase and lyase were present in rumen epithelium. The activities of the enzymes were all lower on a wet weight basis than in liver. Only very slight deacylase activity was detected. 4. Kidney contained acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA lyase and acetoacetyl-CoA deacylase, but only trace amounts of hydroxymethylglutaryl-CoA synthase. 5. Mammary gland contained acetoacetyl-CoA thiolase and some hydroxymethylglutaryl-CoA lyase, but virtually no hydroxymethylglutaryl-CoA synthase or acetoacetyl-CoA deacylase. 6. Since physiologically significant ketogenesis probably occurs solely via the hydroxymethylglutaryl-CoA pathway, it is evident that, of the four tissues examined, such ketogenesis must be restricted to the liver and the rumen epithelium. 7. All the enzymes except hydroxymethylglutaryl-CoA lyase were also assayed in the four tissues derived from cows suffering from bovine lactational ketosis. Ketosis did not cause a statistically significant change in the activity of any of the enzymes measured. 8. Hepatic hydroxymethylglutaryl-CoA synthase and lyase were found to be associated mainly with the particulate fraction, as in the rat. A considerably greater proportion of these enzymes was found to be present in the cytoplasmic fraction from rumen epithelium, although it was not excluded that this was due to mitochondrial damage during homogenization. 9. Appreciable hydroxymethylglutaryl-CoA synthase was also present in epithelium from the dorsal region of the rumen, from the reticulum and from the omasum, but not from the abomasum.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

A radioisotopic method for the determination of acetoacetyl Coenzyme A with 3-hydroxy-3-methylglutaryl Coenzyme A synthase

A simple and sensitive assay for the quantitative determination of acetoacetyl-CoA (AcAc-CoA) in liver and heart is described. The method is based on incorporation of [¹⁴C]acetyl-CoA into acid-stable nonvolatile material in the presence of avian HMG-CoA synthase. The specificity of this procedure for the measurement of AcAc-CoA was demonstrated by pretreating tissue extracts with 3-hydroxyacyl-CoA dehydrogenase or CoA transferase from Escherichia coli to deplete. AcAc-CoA prior to assay. Acid-stable nonvolatile ¹⁴C activity measured in the assay was proportional to the amount of tissue extract added. Satisfactory recovery of AcAc-CoA added at the initial extraction step further validated this procedure. This radioactive assay for acetoacetyl-CoA using a highly purified avian 3-hydroxy-3-methylglutaryl-CoA synthase has the advantages of both extreme specificity for AcAc-CoA as substrate and high sensitivity, facilitating the determination of this metabolite under a variety of physiological conditions.     

A survey of enzymes which generate or use acetoacetyl thioesters in rat liver

Reactions that generate and remove acetoacetyl-CoA and acetoacetate were measured in mitochondria and cytosol of rat liver. The activities surveyed include acetoacetyl-CoA hydrolase, acetoacetyl-glutathione hydrolase, acetoacetyl-CoA:glutathione acyl transferase, 3-ketothiolases I and II, 3-hydroxy-3-methylglutaryl-CoA lyase and synthase, and acetoacetyl-CoA synthetase. Phosphocellulose chromatography shows that cytosol contains at least four acetoacetyl-CoA hydrolase activities, two of which do not coincide with 3-ketothiolases or 3-hydroxy-3-methylglutaryl-CoA lyase, while mitochondria contain at least three acetoacetyl-CoA hydrolase activities that overlap partially or completely with 3-ketothiolases and 3-hydroxy-3-methyl-glutaryl-CoA lyase. Two of the mitochondrial acetoacetyl-CoA hydrolase activities are not found in cytosol. Cytosol contains at least two and mitochondrial extracts at least six acetoacetyl-glutathione hydrolase activities. Mitochondria and cytosol both contain two isozymes of 3-ketoacyl-CoA thiolase (thiolases Ia and Ib). Chain length specificities show that the mitochondrial and cytosolic forms of thiolase Ia differ from each other. We report a new isozyme of 3-ketoacyl-CoA thiolase (thiolase I) in rat liver cytosol.     

Interrelationships between 3-hydroxy-3-methylglutaryl-CoA synthase, acetoacetyl-CoA and ketogenesis

Kinetic and physical approaches have been employed to investigate the binding of acetoacetyl-CoA to hydroxymethylglutaryl-CoA synthase. The enzyme has an apparent Km for acetoacetyl-CoA (0.35 microM) which is more than an order of magnitude lower than the Ki (6--10 microM) measured for substrate inhibition by this metabolite. Hepatic acetoacetyl-CoA concentration, as measured by a sensitive and highly specific radioactive assay appears to be in the 1--10 microM range; the concentration decreases during diabetic ketoacidosis. Total hepatic activity of hydroxymethylglutaryl-CoA synthase and levels of mitochondrial enzyme protein, determined by radioimmunoassay, are not appreciably different in livers from control or ketoacidotic animals. In contrast to the decrease in hepatic acetoacetyl-CoA concentration observed during ketoacidosis, myocardial acetoacetyl-CoA levels are increased by at least tenfold when compared to controls. Elevated acetoacetyl-CoA levels may serve to inhibit fatty acid utilization by the heart. Thus, a consideration of the multiple interactions of acetoacetyl-CoA with the enzymes involved in ketone body production and utilization may be useful in evaluating the metabolic significance of this intermediate.     

Acetoacetate metabolism in rat brain. Development of acetoacetyl-coenzyme A deacylase and 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

1. Data are provided that indicate that the rat brain acetoacetyl-CoA deacylase is almost exclusively mitochondrial. Developmental studies show that this enzyme more than doubles its activity during suckling (0--21 days) and then maintains this activity in adults (approx. 1.1 units/g wet wt.). 2. Kinetic studies (on the acetoacetyl-CoA deacylase) in a purified brain mitochondrial preparation give a Vmax. of 47 nmol/min per mg of protein, and a Km for acetoacetyl-CoA of 5.2 micron and are compatible with substrate inhibition by acetoacetyl-CoA above concentrations of 47 micron. 3. The total brain 3-hydroxy-3-methyl-glutaryl-CoA synthase remains constant in the developing and adult rat brain (approx. 1.2 units/g wet wt.). This enzyme is located in both the mitochondrial and cytosolic fractions. During suckling (0--21 days) the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase represents approx. one-third of the total, but this increases markedly to about 60% of the total in the adult. The cytosolic enzyme correspondingly falls to approx. 40% of the total. 4. The role of the acetoacetyl-CoA deacylase in providing cytosolic acetoacetate for biosynthetic activities in the developing brain is discussed.     

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.     

The purification and some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker''s yeast

1. A purification of 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast is described. This yields a preparation of average specific activity 2.1 units (mumol/min)/mg in which contamination by acetoacetyl-CoA thiolase is less than 0.2%. 2. The molecular weights of 3-hydroxy-3-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase from baker's yeast were determined by gel filtration on Sephadex G-200. The values obtained were 130000 and 190000 respectively. 3. 3-Hydroxy-3-methylglutaryl-CoA synthase is susceptible to irreversible inhibition by a wide variety of alkylating and acylating agents. The time-course of inhibition of the enzyme by some of these, including the active-site-directed inhibitor bromoacetyl-CoA, was studied in the presence and absence of substrates, products and product analogues. Acetyl-CoA, even when present at concentrations as low as 5mum, gives almost complete protection. Other acyl-CoA derivatives give some protection, but only at concentrations 10-30-fold higher. 4. These results are discussed with reference to an ordered reaction pathway in which acetyl-CoA reacts to give a covalent acetyl-enzyme intermediate.     

Effect of phenylmethylsulfonyl fluoride on sterol biosynthesis in 10,000 x g supernatant fraction of rat liver homogenates

Phenylmethylsulfonyl fluoride (PMSF), a reagent commonly employed for the inhibition of serine proteases, has been found to cause significant inhibition of the incorporation of labeled acetate, but not mevalonate, into nonsaponifiable lipid and digitonin-precipitable sterols in the 10,000 X g supernatant fraction of rat liver homogenate preparations. In two experiments, the extent of inhibition of the synthesis of digitonin-precipitable sterols from acetate by PMSF at 1 mM was 81 and 65%. PMSF inhibited the synthesis of nonsaponifiable lipid from acetate at concentrations as low as 0.1 microM. Preincubation of the 10,000 X g supernatant fraction of rat liver homogenates with PMSF (1 mM) resulted in a significant reduction of the activities of acetate thiokinase and 3-hydroxy-3-methylglutaric acid (HMG)-CoA synthase, but did not affect the activities of acetoacetyl-CoA thiolase. Preincubation of rat liver microsomes with PMSF (1 mM) caused a 50% reduction in the level of HMG-CoA reductase activity. The combined results indicate that major sites of action of PMSF in the inhibition of sterol biosynthesis from labeled acetate appear to be on the activities of acetate thiokinase, HMG-CoA synthase, and HMG-CoA reductase. Another reagent used to inhibit serine proteases, diisopropylfluorophosphate, had (at a concentration of 1 mM) no effect on the activities of cytosolic acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase.     

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631)

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.     

3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction.

Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.     

A visible wavelength spectrophotometric assay suitable for high-throughput screening of 3-hydroxy-3-methylglutaryl-CoA synthase

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase catalyzes the first physiologically irreversible step in biosynthesis of isoprenoids and sterols from acetyl-CoA. Inhibition of enzyme activity by β-lactone-containing natural products correlates with substantial diminution of sterol synthesis, identifying HMG-CoA synthase as a potential drug target and suggesting that identification of effective inhibitors would be valuable. A visible wavelength spectrophotometric assay for HMG-CoA synthase has been developed. The assay uses dithiobisnitrobenzoic acid (DTNB) to detect coenzyme A (CoASH) release on acetylation of enzyme by the substrate acetyl-CoA, which precedes condensation with acetoacetyl-CoA to form the HMG-CoA product. The assay method takes advantage of the stability of recombinant enzyme in the absence of a reducing agent. It can be scaled down to a 60 μl volume to allow the use of 384-well microplates, facilitating high-throughput screening of compound libraries. Enzyme activity measured in the microplate assay is comparable to values measured by using conventional scale spectrophotometric assays with the DTNB method (412 nm) for CoASH production or by monitoring the use of a second substrate, acetoacetyl-CoA (300 nm). The high-throughput assay method has been successfully used to screen a library of more than 100,000 drug-like compounds and has identified both reversible and irreversible inhibitors of the human enzyme.     

Requirement for the enzymes acetoacetyl coenzyme A synthetase and poly-3-hydroxybutyrate (PHB) synthase for growth of Sinorhizobium meliloti on PHB cycle intermediates

We have identified two Sinorhizobium meliloti chromosomal loci affecting the poly-3-hydroxybutyrate degradation pathway. One locus was identified as the gene acsA, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase. Analysis of the acsA nucleotide sequence revealed that this gene encodes a putative protein with a molecular weight of 72,000 that shows similarity to acetyl-CoA synthetase in other organisms. Acetyl-CoA synthetase activity was not affected in cell extracts of glucose-grown acsA::Tn5 mutants; instead, acetoacetyl-CoA synthetase activity was drastically reduced. These findings suggest that acetoacetyl-CoA synthetase, rather than CoA transferase, activates acetoacetate to acetoacetyl-CoA in the S. meliloti poly-3-hydroxybutyrate cycle. The second locus was identified as phbC, encoding poly-3-hydroxybutyrate synthase, and was found to be required for synthesis of poly-3-hydroxybutyrate deposits.     

3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative.

Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a 'half-of-the-sites' reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.     

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.     

Staphylococcus aureus 3-hydroxy-3-methylglutaryl-CoA synthase: crystal structure and mechanism

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a member of the family of acyl-condensing enzymes, catalyzes the first committed step in the mevalonate pathway and is a potential target for novel antibiotics and cholesterol-lowering agents. The Staphylococcus aureus mvaS gene product (43.2 kDa) was overexpressed in Escherichia coli, purified to homogeneity, and shown biochemically to be an HMG-CoA synthase. The crystal structure of the full-length enzyme was determined at 2.0-A resolution, representing the first structure of an HMG-CoA synthase from any organism. HMG-CoA synthase forms a homodimer. The monomer, however, contains an important core structure of two similar alpha/beta motifs, a fold that is completely conserved among acyl-condensing enzymes. This common fold provides a scaffold for a catalytic triad made up of Cys, His, and Asn required by these enzymes. In addition, a crystal structure of HMG-CoA synthase with acetoacetyl-CoA was determined at 2.5-A resolution. Together, these structures provide the structural basis for an understanding of the mechanism of HMG-CoA synthase.     

The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker''s yeast

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have K(i) values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of K(i) with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).     

Metabolic engineering of Alcaligenes eutrophus through the transformation of cloned phbCAB genes for the investigation of the regulatory mechanism of polyhydroxyalkanoate biosynthesis

The regulatory mechanisms of the biosynthesis of in vivo poly-beta-hydroxybutyrate [PHB] and poly(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] of Alcaligenes eutrophus were investigated by using various transformants with enzyme activities that were modified through the transformation of cloned phbCAB genes. The biosynthesis rates of PHB and P(3HB-3HV) were controlled by beta-ketothiolase and acetoacetyl-CoA reductase, and especially by beta-ketothiolase condensing acetyl-CoA or propionyl-CoA. The contents of PHB and P(3HB-3HV) were controlled by PHB synthase, polymerizing 3-hydroxybutyrate to PHB or 3-hydroxybutyrate and 3-hydroxyvalerate to P(3HB-3HV). The molar fraction of 3-hydroxyvalerate in P(3HB-3HV) was also closely connected with PHB synthase. This may be due to the accelerated polymerization between 3-HB from glycolysis pathway and 3-HV converted from propionate supplied as precursor. Enforced beta-ketothiolase and acetoacetyl-CoA reductase to PHB synthase tended to enlarge the size of the PHB and P(3HB-3HV) granules, however, higher activity ratio of PHB synthase to beta-ketothiolase and acetoacetyl-CoA reductase than parent strain tended to induce the number of granules.