Mechanism and specificity of succinyl-CoA:3-ketoacid coenzyme A transferase.

(a) The reactivity of substituted acetates as substrates for CoA transferase increases sharply with increasing basicity and exhibits a slope of 1.0 in a plot of either log kappacat or log (kappacat/Km) against pKa (betanuc = 1.0). This result shows that the catalyzed reaction, which involves both carboxylate activation and leaving group transfer, does not proceed through a fully concerted reaction mechanism in the rate-determining step. The result is consistent with a stepwise reaction mechanism that proceeds through an anhydride intermediate. (b) Equilibrium constants for thiol ester formation, either bound to the enzyme or free in solution, show the same dependence on the basicity of carboxylate ions (betaeq = 1.0) and are independent of acidity when expressed in terms of the carboxylic acid. Thus, the polar environment around substituents on the acyl group is the same for carboxylic acids, thiol esters, and oxygen esters. (c) The interaction of the terminal CH3CO group of acetoacetate with the active site causes a 200,000-fold increase in kappacat/Km, corresponding to a decrease in delta G++ OF 7.2 kcal/mol compared with an unsubstituted acid of the same pK. The binding energy of the coenzyme A moiety of the substrate is utilized to interact with the active site and cause a 10(4) to 10(6)-fold increase in kappacat, corresponding to a decrease in delta G++ of 6 to 9 kcal/mol, compared with fragments of the coenzyme A moiety added separatly or together. (d) The exchange of labeled coenzyme A into acyl-CoA substrates was found to be greater than or equal to 10(5) slower than substrate turnover.     

Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28 or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys 28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein was unexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a different conformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On the basis of these results and the studies of the structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17 degrees coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate or acetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has led to the proposal of a new mechanism for catalysis by SCOT.     

Formation of acetoacetate from 3-hydroxy-3-methylglutarate by rat liver and isolation of a mitochondrial coenzyme A-transferase activity involved

1. Formation of acetoacetate from 3-hydroxy-3-methylglutarate was observed in the perfused rat liver. Production of 3.5mumol of acetoacetate/h per g of tissue was obtained. 2. Formation of acetoacetate was catalysed mainly by the mitochondrial fraction of the homogenized liver, at a rate of 62nmol/h per mg of protein. 3. Experiments with hydroxy-[3-(14)C]methylglutarate demonstrated that the acetoacetate formed was derived mainly from this compound. 4. A mitochondrial transferase activity catalysing the transfer of a CoA molecule from succinyl-CoA (3-carboxypropionyl-CoA) to hydroxymethylglutarate was shown. The K(m) value for hydroxymethylglutarate was 5x10(-3)m.     

Formation and utilization of 3-hydroxy-3-methylglutarate in liver mitochondria of starved and streptozotocin-diabetic rats.

Kidney and liver mitochondria of rat, rabbit and guinea pig are able to transform 3-hydroxy-3-methylglutarate into acetoacetate, whereas ox liver mitochondria and rat mitochondria of heart, diaphragm and brain do not exhibit such an activity. Starvation and streptozotocin treatment decreases the formation of acetoacetate from 3-hydroxy-3-methylglutarate. Addition of acetoacetate and succinate to the incubation media of mitochondria results in a decrease in the transformation of 3-hydroxy-3-methylglutarate into acetoacetate. A 3-hydroxy-3-methylglutaryl-CoA hydrolase is present in rat liver mitochondria; the activity does not show appreciable changes after starvation or streptozotocin treatment.     

Solubilization and partial purification of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

This paper describes an effective method for the solubilization of microsomal HMG-CoA reductase from rat liver. Exposing the microsomes to a freeze-thaw treatment solubilized 80% of the microsomal reductase activity. Subsequently, a 25-fold purification has led to an enzyme preparation with a specific activity of 10–14 nmoles MVA per min per mg of protein and an increased stability.     

Isolation and immunological characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) has been isolated from human liver utilizing HMG-CoA affinity chromatography. The apparent monomer molecular weight of purified human HMG-CoA reductase by SDS-gel electrophoresis was 53,000, and the oligomeric molecular weight determined by sucrose density centrifugation was 104,000. A monospecific antibody prepared against rat liver HMG-CoA reductase inhibited the enzymic activity of microsomal and purified human liver enzyme and formed a single immunoprecipitin line by radial immunodiffusion. These results represent the initial isolation and characterization of human liver HMG-CoA reductase.     

Partial purification and characterization of 3-hydroxy-3-methylglutaryl coenzyme a reductase from the leaves of guayule (Parthenium argentatum)

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been isolated and was partially purified from the leaves of Parthenium argentatum. The enzyme was found to be associated both with the cytosol and the chloroplasts. Ten mM dithiothreitol was essential to prevent loss of activity. Optimum activities of cytosolic and chloroplastic fractions were observed at pH 7.0 and 7.5 respectively. Preincubation of the reaction mixtures with CoA, acetyl-CoA, σ-phenanthroline and iodoacetamide resulted in the progressive loss of enzyme activity. 3-Hydroxybutyrate and mevalonate also inhibited the enzyme. The Michaelis constants of the enzyme for HMG-CoA and NADPH were 0.25 and 0.31 mM respectively for the cytosolic enzyme, while those for the chloroplastic enzyme were 0.018 and 0.42 mM respectively. Inhibition studies indicated that hydroxybutyrate was a competitive inhibitor with respect to HMG-CoA. The inhibition of mevalonate was competitive with HMG-CoA and non-competitive with NADPH.     

Partial purification and characterization of succinyl-CoA synthetase from Saccharomyces cerevisiae

Succinyl-CoA synthetase from Saccharomyces cerevisiae was partially purified (20-fold) with a yield of 44%. The Michaelis-Menten constants were determined: Km (succinate) = 17 mM; Km (ATP) = 0.13 mM; Km (CoA) = 0.03 mM. The succinyl-CoA synthetase has a molecular weight of about 80000 dalton (as determined by polyacrylamide gradient gel electrophoresis). The pH optimum is at 6.0. During fermentation the activity of succinyl-CoA synthetase is lower than in aerobically grown yeast cells. The presence of succinyl-CoA synthetase in fermenting yeasts may be regarded as an indication for the oxidative formation of succinate. In fermenting yeast cells succinyl-CoA synthetase is repressed by glucose if ammonium sulphate serves as nitrogen source. This catabolite repression is not observed with disaccharides or when amino acids are used as nitrogen source.     

Further purification and characterization of the DNA 3'-phosphatase from rat-liver chromatin which is also a polynucleotide 5'-hydroxyl kinase

The DNA 3'-phosphatase activity of rat-liver chromatin has been purified. A DNA 5'-hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5'-hydroxyl kinase and a 3'-phosphatase. An additional argument is that ATP, which does not influence the rate of the 3'-phosphatase reaction but which is a cosubstrate of the 5'-hydroxyl kinase, protects the 3'-phosphatase activity against thermal denaturation and trypsin digestion. The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion. The rates of 3'-dephosphorylation and 5'-phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3'-phosphatase has no action on 3'-monodeoxynucleotide, but it hydrolyzes the 3'-phosphate in dinucleotides. The Km of the 3'-phosphatase is 0.548 microM. The Km (5'-OH) and Km (ATP) of the 5'-hydroxyl kinase are about 3.9 microM and 0.69 microM respectively. The chromatin enzyme is unable to hydrolyze 3'-phosphoglycolate ends in DNA.     

Purification and characterization of avian liver 3-hydroxy-3-methylglutaryl coenzyme A lyase

3-Hydroxy-3-methylglutaryl-CoA lyase has been purified to homogeneity from avian liver mitochondria. Affinity chromatography of a partially purified preparation on agarose hexane 3',5'-ADP produces enzyme of high specific activity (351 units/mg). A total purification of 1750-fold over the mitochondrial matrix fraction is achieved. The purified enzyme is stable when stored in 30% glycerol with millimolar levels of dithiothreitol. Divalent cations (e.g. Mg2+, Mn2+) and thiol-protecting agents stimulate enzyme activity under assay conditions. The enzyme binds hydroxymethylglutaryl-CoA with a Km = 8 microM. Optimal enzyme activity, measured at pH = 8.9, is 7-fold higher than activity at physiological pH. The apparent molecular weight of the native enzyme, estimated by gel filtration on Sephadex G-100, is approximately 49,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggests that the enzyme is a dimer, composed of 27,000-dalton subunits. Assuming one active site per subunit, a turnover number of 158 s-1 (pH 8.2; 30 degrees C) is calculated. Antibodies have been prepared against homogeneous hydroxymethylglutaryl-CoA lyase. Ouchterlony double diffusion patterns verify the homogeneity of the preparation. Incubation of enzyme with antiserum results in virtually complete inhibition of enzyme activity.     

Nitration of tryptophan 372 in succinyl-CoA:3-ketoacid CoA transferase during aging in rat heart mitochondria

The main objective of this study was to test the hypothesis that in vivo post-translational modifications in proteins, induced by the endogenously generated reactive oxygen and nitrogen molecules, can alter protein function and thereby have an effect on metabolic pathways during the aging process. Succinyl-CoA:3-ketoacid coenzyme A transferase (SCOT), the mitochondrial enzyme involved in the breakdown of ketone bodies in the extrahepatic tissues, was identified in rat heart to undergo age-associated increase in a novel, nitro-hydroxy, addition to tryptophan 372, located in close proximity ( approximately 10 A) of the enzyme active site. Between 4 and 24 months of age, the molar content of nitration was more than doubled while specific enzyme activity increased significantly. The amount of SCOT protein, however, remained unchanged. In vitro treatment of heart mitochondrial soluble proteins with relatively low concentrations of peroxynitrite enhanced the nitration as well as specific activity of SCOT. Results of this study identify tryptophan to be a specific target of nitration in vivo, for the first time. We hypothesize that increases in tryptophan nitration of SCOT and catalytic activity constitute a plausible mechanism for the age-related metabolic shift toward enhanced ketone body consumption as an alternative source of energy supply in the heart.     

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acid coenzyme A transferase from rat liver mitochondria.

Substrate specificity of a dicarboxyl-CoA: dicarboxylic acids coenzyme A transferase purified from rat liver mitochondria was assayed. In addition to the previously identified substrates succinyl-CoA, 3-hydroxy-3-methylglutaryl-CoA and malonyl-CoA (Francesconi et al.(1989) Biochim. Biophys. Acta, 999, 163-170) also methylmalonyl-CoA, glutaryl-CoA and adipyl-CoA acted as enzyme substrates, with the latter thioester showing the highest apparent affinity. All corresponding dicarboxylic acids, but not oxaloacetic, citric, alpha-ketoglutaric, malic, fumaric and glutamic acids, acted as coenzyme A acceptor substrates. None of the tested monocarboxylic acids, or the corresponding coenzyme A esters, were enzymatically transformed by the here described coenzyme A transferase.     

Phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent M_r of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-³²P]ATP produces a unique band containing ³²P bound to protein which migrates at the same R_f as the reductase subunit. Incubation of ³²P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound ³²P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, ³²P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one ³²P-labeled phosphopeptide with the same R_f as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed.     

Isolation and characterization of a cDNA encoding a 3-hydroxy-3-methylglutaryl coenzyme A reductase from Nicotiana sylvestris

A cDNA library from freshly isolated mesophyll protoplasts of Nicotiana sylvestris was differentially screened using cDNAs from leaves, leaf strips submitted to the same stress as protoplasts during the isolation procedure, and cell suspension cultures. One of the selected clones (6P2) was found to encode a putative polypeptide highly homologous to previously characterized 3-hydroxy-3-methylglutaryl coenzyme A reductases. The C-terminal region of the polypeptide was highly conserved whereas its N-terminal region including the trans-membrane domains and the linker was more variable. Apart from protoplasts, the 6P2 gene was found to be expressed in apexes, anthers, roots, and in stressed leaf strips after 24h of culture, during the hypersensitive reaction to viral infection and after HgCl₂ treatment. This pattern of expression is consistent with a role in plant defence mechanisms.     

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat.     

Cloning and functional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania somnifera: an important medicinal plant

Withania somnifera (L.) Dunal is one of the most valuable medicinal plants synthesizing a large number of pharmacologically active secondary metabolites known as withanolides, the C₂₈-steroidal lactones derived from triterpenoids. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the biosynthetic pathway and genes responsible for biosynthesis of these compounds. In this study, we have characterized the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; EC 1.1.1.34) catalyzing the key regulatory step of the isoprenoid biosynthesis. The 1,728-bp full-length cDNA of Withania HMGR (WsHMGR) encodes a polypeptide of 575 amino acids. The amino acid sequence homology and phylogenetic analysis suggest that WsHMGR has typical structural features of other known plant HMGRs. The relative expression analysis suggests that WsHMGR expression varies in different tissues as well as chemotypes and is significantly elevated in response to exposure to salicylic acid, methyl jasmonate, and mechanical injury. The functional color assay in Escherichia coli showed that WsHMGR could accelerate the biosynthesis of carotenoids, establishing that WsHMGR encoded a functional protein and may play a catalytic role by its positive influence in isoprenoid biosynthesis.