Isolation and expression of HMG-CoA synthase and HMG-CoA reductase genes in different development stages, tissues and treatments of the Chinese white pine beetle, Dendroctonus armandi (Curculionidae: Scolytinae).

We isolated two full-length cDNAs encoding 3-hydroxy-3-methyl-glutaryl coenzyme A synthase (HMG-S) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R) from the Chinese white pine beetle (Dendroctonus armandi), and carried out some bioinformatic analysis on the full-length nucleic acid sequences and deduced amino acid sequences. Differential expression of the DaHMG-S and DaHMG-R genes was observed between sexes (emerged adults), and within these significant differences among development stage, tissue distribution, fed on phloem of Pinus armandi and topically applied juvenile hormone (JH) III. Increase of DaHMG-S and DaHMG-R mRNA levels in males suggested that they may play a role in mevalonate pathway. Information from the present study might contribute to understanding the relationship between D. armandi and its semiochemical production.

A structural limitation on enzyme activity: the case of HMG-CoA synthase.

Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine. Subsequent enzymatic studies showed that the overall reaction rate of the enzyme was increased 140-fold. An X-ray crystallographic study of the Ala110Gly mutant enzyme demonstrated unanticipated adjustments in the active site that resulted in additional stabilization of all three steps of the reaction pathway. The rates of acetylation and hydrolysis of the mutant enzyme increased because the amide nitrogen of Ser308 shifts 0.4 A toward the catalytic cysteine residue. This motion positions the nitrogen to better stabilize the intermediate negative charge that develops on the carbonyl oxygen of the acetyl group during both the formation of the acyl-enzyme intermediate and its hydrolysis. In addition, the hydroxyl of Ser308 rotates 120 degrees to a position where it is able to stabilize the carbanion intermediate formed by the methyl group of the acetyl-S-enzyme during its condensation with acetoacetyl-CoA.

3-Hydroxy-3-methylglutaryl-CoA synthase: participation of invariant acidic residues in formation of the acetyl-S-enzyme reaction intermediate.

Inactivation of HMG-CoA synthase by a carboxyl-directed reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in a concentration-dependent and substrate-protectable manner suggested that the active site contains reactive acidic amino acids. This observation prompted functional evaluation of 11 invariant acidic amino acids by site-directed mutagenesis. Characterization of the isolated synthase variants' ability to catalyze overall and partial reactions identified three mutant synthases (D99A, D159A, and D203A) that exhibit significant diminution of k(cat) for the overall reaction (10(2)-, 10(3)-, and 10(4)-fold decreases, respectively). D99A, D159A, and D203A form the acetyl-S-enzyme intermediate very slowly (0.0025, 0.0026, 0.0015 U/mg, respectively, measured at pH 7. 0 and 22 degrees C) as compared to the wild-type synthase (1.59 U/mg), where intermediate formation approaches rate-limiting status. Differences in substrate saturation do not account for impaired activities or rates of intermediate formation. The structural integrity of the purified mutants' active sites is demonstrated by their abilities to bind a spin-labeled acyl-CoA analogue (R.CoA) with affinities and stoichiometries comparable to values measured for wild-type synthase. The impact of three distinct amino acids on reaction intermediate formation supports a mechanism of acetyl-S-enzyme formation that probably requires formation and directed collapse of a tetrahedral adduct. (18)O-induced shift of the (13)C NMR signal of (13)C acetyl-S-enzyme demonstrates that an analogous tetrahedral species is produced upon solvent exchange with the acetyl-S-enzyme. Partial discrimination between the functions of D99, D159, and D203 becomes possible based on the observation that D159A and D203A synthases exhibit retarded kinetics of solvent (18)O exchange while D99A fails to support (18)O exchange.

Isolation and endocrine regulation of an HMG-CoA synthase cDNA from the male Jeffrey pine beetle, Dendroctonus jeffreyi (Coleoptera: Scolytidae).

We have isolated a full length 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) cDNA from the male Jeffrey pine beetle, Dendroctonus jeffreyi Hopkins, and studied the effects of topical applications of juvenile hormone III (JH III) on its expression. The predicted translation product of this apparently single copy gene has 63% and 58% identity with HMG-S1 and HMG-S2 from Blattella germanica (L.), and 61% identity with Drosophila melanogaster Hmgs. HMG-S transcript levels remain uniformly low in JH III-treated and control D. jeffreyi females, but are induced approximately 2.5- to 5-fold in JH III-treated males. JH III causes a dose- and time-dependent increase in HMG-S transcripts in the male metathoracic-abdominal region. Since monoterpenoid pheromone precursor synthesis and HMG-CoA reductase expression are under the control of JH III in the metathorax of Ips bark beetles, the observed HMG-S expression pattern suggests that the isoprenoid pathway is similarly important for semiochemical production in D. jeffreyi.

Catalytic properties of recombinant 3-hydroxy-3-methylglutaryl coenzyme A synthase-1 from Blattella germanica.

Blattella germanica is the first organism in which two cytosolic HMG-CoA synthase genes have been described: HMGS-1 (Martínez-González et al., 1993b) and HMGS-2 (Buesa et al., 1994). The HMGS-1 gene showed special features, which led us to characterize the kinetic properties of the enzyme it encodes. Here we report the expression of recombinant HMGS-1, the protocol of enzyme purification, and the measurement of kinetic parameters. The K(m) for acetyl-CoA is 15.2 microM and the Ki for the other substrate, acetoacetyl-CoA, is 1.26 microM, both similar to that of yeast, ox, and chicken liver enzymes; the Vmax of HMGS-1 measured in this paper is 66 mU, which is the lowest Vmax of the HMG-CoA synthases reported to date.

Avian cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase: evaluation of the role of cysteines in reaction chemistry.

The pH dependence of avian cytosolic HMG-CoA synthase activity is fit by a titration curve with a pK = 8.6. The observation of optimal activity at alkaline pH and the insensitivity of pK to divalent cation concentration suggest that the pK reflects ionization of an amino-acid side chain (e.g., cysteinyl sulfhydryl) rather than substrate enolization. Upon reaction of 3-chloropropionyl-CoA with HMG-CoA synthase C129S, an enzyme variant lacking the sulfhydryl group normally targeted by this mechanism-based inhibitor, stoichiometric modification occurs. Amino-acid analysis indicates that cysteine is the principal target in C129S enzyme, demonstrating the presence of a second reactive cysteine within this enzyme. To test whether another cysteine functions in reaction chemistry, conserved cysteines were identified by sequence homology analysis. Five cysteine residues (C59, C69, C224, C232, C268), invariant in the nine sequences available for various eukaryotic HMG-CoA synthase isozymes, were individually replaced by alanine in a series of mutant enzymes. Kinetic analyses of the isolated mutant HMG-CoA synthases indicate that none of these is crucial to the chemistry that results in production of HMG-CoA. These results further distinguish the HMG-CoA synthase reaction from the related condensation of acyl-CoA substrates catalyzed by beta-ketothiolase.

A novel assay for cytosolic 3-hydroxy-3-methylglutaryl-coenzyme A synthase activity using reversed-phase ion-pair chromatography: demonstration that Lifibrol (K12.148) modulates the enzyme activity.

Cytosolic HMG-CoA synthase and microsomal 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase catalyze two sequential steps in the mevalonate pathway. Both enzymes are negatively regulated by cholesterol. Cytosolic HMG-CoA synthase is responsible for the generation of HMG-CoA from acetyl-CoA and acetoacetyl-CoA). We have developed a new method to determine HMG-CoA synthase activity. In this assay, HMG-CoA is formed from acetoacetyl-CoA and labeled acetyl-CoA. The HMG-CoA product is isolated from the reaction mixture by means of reversed-phase ion-pair chromatography. The recovery of the product was always greater than 90%. The average within-batch coefficient of variation for HMG-CoA synthase activity was 5.1%. Using the new assay, we demonstrate that Lifibrol (K12.148), a new hypolipidemic compound, inhibits HMG-CoA synthase. Because our assay is accurate and precise it may become useful in future studies on the regulation and the pharmacological modulation of cytosolic HMG-CoA synthase.

Human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase: expression, purification, and characterization of recombinant wild-type and Cys129 mutant enzymes.

A cDNA for the human cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (EC 4.1.3.5) was subcloned and expressed from a T7-based vector in Escherichia coli. The over-produced enzyme was purified using a three-step protocol that generated 20 to 30 mg protein/liter cell culture. The physical and catalytic properties of the recombinant synthase are similar to those reported for the nonrecombinant enzymes from chicken liver [Clinkenbeard et al. (1975a) J. Biol. Chem. 250, 3124-3135] and rat liver [Mehrabian et al. (1986) J. Biol. Chem. 261, 16249-16255]. Mutation of Cys129 to serine or alanine destroys HMG-CoA synthase activity by disrupting the first catalytic step in HMG-CoA synthesis, enzyme acetylation by acetyl coenzyme A. Furthermore, unlike the wild-type enzyme, neither mutant was capable of covalent modification by the beta-lactone inhibitor, L-659,699 [Greenspan et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7488-7492]. Kinetic analysis of the inhibition by L-659,699 revealed that this compound is a potent inhibitor of the recombinant human synthase, with an inhibition constant of 53.7 nM and an inactivation rate constant of 1.06 min-1.

Regulation of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase protein by starvation, fat feeding, and diabetes.

We have determined the levels of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase under different metabolic situations to examine its potential role as a regulatory protein in the ketogenic pathway. We used specific antibodies directed against a peptide of the amino acid sequence of the protein as deduced from the cDNA sequence. The amount of mitochondrial HMG-CoA synthase protein rapidly increased in response to cyclic AMP, dexamethasone, starvation, fat feeding, and diabetes, whereas it was decreased by insulin and refeeding. Insulin was also able to counteract the increase in mitochondrial HMG-CoA synthase levels observed under the diabetic condition. Furthermore, the finding that quantitative changes in HMG-CoA synthase protein were less marked than those in the corresponding mRNA in starved and diabetic rats suggests either translational control or increased degradation of either mRNA or protein. All these results indicate that mitochondrial HMG-CoA synthase is a regulatory element in the ketogenic process.

Avian 3-hydroxy-3-methylglutaryl-CoA synthase. Characterization of a recombinant cholesterogenic isozyme and demonstration of the requirement for a sulfhydryl functionality in formation of the acetyl-enzyme reaction intermediate.

cDNA encoding avian liver hydroxymethylglutaryl-CoA synthase has been cloned into a pET vector, and the resulting expression plasmid has been used to transform Escherichia coli BL21 (DE3). Heterologous expression of hydroxymethylglutaryl-CoA synthase occurs upon growth of this bacterial strain in the presence of isopropyl-1-thio-beta-D-galactopyranoside, with the target enzyme representing over 20% of total cellular protein. Recombinant enzyme is soluble and stable in crude E. coli extracts, facilitating its isolation in homogeneous form. With respect to specific activity, acylation stoichiometry, Km,Ac-CoA, and binding of a spin-labeled substrate analog, the recombinant enzyme is equivalent to avian enzyme, suggesting its utility for mechanistic and structural studies. Our earlier prediction that this avian cDNA encodes the cholesterogenic cytosolic isozyme is supported by a series of experimental observations. Upon SDS-polyacrylamide gel electrophoresis, the recombinant synthase exhibits mobility in agreement with the 57.6-kDa deduced molecular mass, which exceeds the 53-kDa estimate and experimental observation for the ketogenic mitochondrial isozyme. Activity of the recombinant synthase is stimulated by Mg2+, as predicted for the cholesterogenic cytosolic isozyme and in contrast to the inhibition observed for the mitochondrial isozyme. Although antibody prepared against avian mitochondrial synthase effectively detects both avian mitochondrial and recombinant synthases on Western blots, antibody prepared against rodent cytosolic synthase discriminates between the two proteins, sensitively detecting recombinant enzyme while reacting poorly with authentic mitochondrial enzyme. Directed mutagenesis of the recombinant synthase has been performed to produce a C129S variant, in which the sulfhydryl previously implicated in formation of the acetyl-S-enzyme reaction intermediate is replaced by a hydroxyl group. EPR measurements on the binary C129S-spin-labeled acyl-CoA complex demonstrate that the mutant's substrate binding site is unperturbed in comparison with wild-type protein. These data illustrate the utility of spin-labeled substrate analogs as tools to stringently evaluate the structural integrity of engineered proteins. C129S is catalytically inactive (10(5)-fold decrease in kcat) despite retaining the ability to form noncovalent complexes with acetyl-CoA or a spin-labeled acetyl-CoA analog. The demonstrated failure of C129S to form a covalent acyl-O-enzyme species accounts for these observations; data derived from experiments performed with a C129G mutant confirm this conclusion. These results distinguish hydroxymethylglutaryl-CoA synthase from beta-ketoacyl thiolase.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase and cholesterol biosynthesis by beta-lactone inhibitors and binding of these inhibitors to the enzyme.

The beta-lactones L-659,699 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2- oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic acid) and its radioactive derivative 3H-L-668,411 (the 2,3-ditritiated methyl ester of L-659,699) inhibited a partially purified preparation of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase with an IC50 of 0.1 microM. These compounds were also found to inhibit the incorporation of [14C]acetate into sterols in cultured Hep G2 cells with an IC50 of 3 microM. New kinetic evidence indicated that inhibition of the isolated enzyme was irreversible. In contrast, sterol biosynthesis in cultured Hep G2 cells was rapidly restored upon removal of the compound from the medium of inhibited cultures, suggesting reversibility of inhibition in the cells. Radioactivity was found to be associated with a single cytoplasmic protein by SDS/PAGE of the cytoplasm of Hep G2 cells after incubation of the cells with the inhibitor 3H-L-668,411. This protein was identified as cytoplasmic HMG-CoA synthase. Binding of the radioactive compound to the enzyme was decreased with time if the radioactive inhibitor was removed from the medium. Exposure of a gel containing the radioactive enzyme-inhibitor complex to neutral hydroxylamine also resulted in a loss of radioactivity from the gel. The purified rat liver enzyme reacted with the 3H-ligand to form a stable enzyme-inhibitor complex which could be isolated by h.p.l.c. Radioactivity was also subsequently lost from this complex when it was incubated with neutral hydroxylamine. Incorporation of [14C]acetate into cholesterol in mouse liver was inhibited in a reversible manner after oral administration of the beta-lactone inhibitor. These studies, as well as the kinetic evidence presented, suggest that the beta-lactone inhibitors acylate HMG-CoA synthase in a reaction which appears to be irreversible in vitro, but is easily reversed in cultured cells and in animals.

Regulation of cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase mRNA levels by L-tri-iodothyronine.

In hypophysectomized and thyroidectomized rats, cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase activity, immunoreactive protein and mRNA levels were all considerably decreased. Administration of L-tri-iodothyronine (T3) resulted in a large increase in all three in hypophysectomized rats and in only a 2-fold increase (reaching the values of control rats) in thyroidectomized rats.

Diurnal rhythm of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase.

Rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase exhibits a diurnal rhythm of enzyme activity which coincides with the diurnal rhythm of HMG-CoA synthase protein. The peaks of activity and protein (determined by SDS/PAGE and immunoblotting) both occur at D10 (the tenth hour of the daily 12 h dark cycle). The peak of mRNA levels (measured by slot-blot hybridization of liver RNA) is slightly advanced with respect to that of protein, by about 4 h, and shows a maximum at D6. Cytosolic HMG-CoA synthase activity and protein in rats fed on a normal diet were approx. 2-fold higher during the peak at D10 than in the nadir at D2. HMG-CoA synthase mRNA levels were approx. 4-fold higher during the peak at D6 than in the nadir at D2. These results point to a transcriptional and translational regulation of the cytosolic HMG-CoA synthase.

Aspects related to mevalonate biosynthesis in plants.

We purified and characterized a membrane-associated enzyme system from radish (Raphanus sativus L.) that is capable of converting acetyl-CoA into 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). The enzyme system apparently comprises acetoacetyl-CoA thiolase (EC 2.3.1.9) and HMG-CoA synthase (EC 4.1.3.5). Its activity in vitro can be strongly stimulated by FeII. When ferrous ions are applied chelated with ethylenediaminetetraacetate, citrate or adenosine 5-triphosphate (ATP), the stimulation is further increased. Stimulation is due to a higher catalytic efficiency as indicated by an increase in Vmax, whereas the affinity of the enzyme towards acetyl-CoA remains constant (Km = 6 micro M). A considerable portion of HMG-CoA lyase activity is associated with the same membranes. HMG-CoA lyase (EC 4.1.3.4) is also solubilized and partially co-purified. Its activity requires comparatively high concentrations of Mg2+. The conversion of HMG-CoA to mevalonic acid is catalyzed by HMG-CoA reductase (EC 1.1.1.34) that is associated with the same membranes. By cDNA encoding the Arabidopsis HMG-CoA reductase, we isolated a corresponding gene from a cDNA library newly established from etiolated radish seedlings. This full-length cDNA, referred to as lambda cRS3, encodes a polypeptide 583 amino acids with a molecular mass of about 63 kDa. The hydropathy profile suggests the presence of two hydrophobic membrane-spanning domains within the N-terminal 165 amino acids. The carboxy-terminal part, where the catalytic site resides, is highly conserved in all eukaryotic HMG-CoA reductase genes sequenced so far.

3-Hydroxy-3-methylglutaryldithio-coenzyme A: a potent inhibitor of Pseudomonas mevalonii HMG-CoA reductase.

3-Hydroxy-3-methyl-1-thionoglutaryl-coenzyme A, a dithioester analog of 3-hydroxy-3-methylglutaryl-CoA, has been enzymatically synthesized using the HMG-CoA synthase catalyzed condensation of acetyl-CoA with 3-oxo-1-thionobutyryl-CoA. HMGdithio-CoA is a potent inhibitor of Pseudomonas mevalonii HMG-CoA reductase. Inhibition was mainly competitive with respect to HMG-CoA with a Kis of 0.086 +/- .01 microM and noncompetitive with respect to NADH with a Kis of 3.7 +/- 1.5 microM and a Kii of 0.65 +/- .05 microM in the presence of 110 microM (R.S)-HMG-CoA.

Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699.

A beta-lactone isolated from Fusarium sp. has been shown to be a potent specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acetylating), EC, 4.1.3.5] from rat liver. The structure of this beta-lactone, termed L-659,699, is (E,E)-11-[3-(hydroxy-methyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4 - undecadienenoic acid. A partially purified preparation of cytoplasmic HMG-CoA synthase from rat liver was inhibited by L-659,699 with an IC50 of 0.12 microM. The enzyme HMG-CoA reductase, beta-ketoacyl-CoA thiolase, acetoacetyl-CoA synthetase, and fatty acid synthase were not inhibited to any extent by this compound. In cultured Hep G2 cells, the compound inhibited the incorporation of [14C]acetate into sterols with an IC50 of 6 microM, while incorporation of [3H]mevalonate into sterols in these cells was not affected. The activity of HMG-CoA reductase in the cultured Hep G2 cells was induced in a dose-dependent manner by incubation with L-659,699. A 37-fold increase in reductase was observed after a 24-hr incubation with 62 microM L-659,699. The effect of a number of analogs of L-659,699 on HMG-CoA synthase is also discussed.

Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A synthase and the chromosomal localization of the human gene.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was purified to homogeneity from rat liver cytoplasm. The active enzyme is a dimer composed of identical subunits of Mr = 53,000. The amino acid composition and the NH2-terminal sequence are presented. Partial cDNA clones for the enzyme were isolated by screening of a rat liver lambda gt11 expression library with antibodies raised against the purified protein. The identity of the clones was confirmed by hybrid selection and translation. When rats were fed diets supplemented with cholesterol, cholestyramine, or cholestyramine plus mevinolin, the hepatic protein mass of cytoplasmic synthase, as determined by immunoblotting, was 25, 160, and 1100%, respectively, of the mass observed in rats fed normal chow. Comparable changes in enzyme activity were observed. Approximately 9-fold increases in both HMG-CoA synthase mRNA mass and synthase mRNA activity were observed when control diets were supplemented with cholestyramine and mevinolin. When rats were fed these two drugs and then given mevalonolactone by stomach intubation, there was a 5-fold decrease of synthase mRNA within 3 h. These results indicate that cytoplasmic synthase regulation occurs primarily at the level of mRNA. This regulation is rapid and coordinate with that observed for HMG-CoA reductase. The chromosomal localization of human HMG-CoA synthase was determined by examining a panel of human-mouse somatic cell hybrids with the rat cDNA probe. Interestingly, the synthase gene resides on human chromosome 5, which has previously been shown to contain the gene for HMG-CoA reductase. Regional mapping, performed by examination of a series of chromosome 5 deletion mutants and by in situ hybridization to human chromosomes indicates that the two genes are not tightly clustered.

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA.

We here report the isolation and nucleotide sequencing of a full-length 3.3-kilobase cDNA for the cytoplasmic form of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a regulated enzyme in the cholesterol biosynthetic pathway. The cDNA was isolated from UT-1 cells, a compactin-resistant line of Chinese hamster ovary cells. UT-1 cells produce large amounts of mRNA for HMG-CoA synthase and the next enzyme in the pathway, HMG-CoA reductase, as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. The identity of the cDNA for HMG-CoA synthase was confirmed through comparison of the NH2-terminal amino acid sequence predicted from the cDNA with that determined chemically from the purified enzyme. Anti-peptide antibodies directed against the amino acid sequence predicted from the cDNA precipitated HMG-CoA synthase activity from liver cytoplasm. The feeding of cholesterol to hamsters led to a decrease of more than 85% in the amount of mRNA for HMG-CoA synthase and HMG-CoA reductase in hamster liver. These data indicate that the mRNAs for cytoplasmic HMG-CoA synthase and for HMG-CoA reductase, two sequential enzymes in the cholesterol biosynthetic pathway, are coordinately regulated by cholesterol.

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.