Characterization of Citrate Synthase from Geobacter sulfurreducens and Evidence for a Family of Citrate Synthases Similar to Those of Eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (k_cat = 8.3 s⁻¹; K_m = 14.1 and 4.3 μM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (K_i = 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K⁺ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

Guanosine triphosphate and colchicine affect the activity and the polymeric state of acetyl-CoA carboxylase

Acetyl-CoA carboxylase was purified 300-fold from rat liver, in the absence of added citrate, by precipitation from an 18,000g supernatant in the presence of Triton X-100 at 105,000g and 20 °C, followed by chromatography on phosphocellulose. Acetyl-CoA carboxylase activity in this preparation was activated by preincubation with GTP (0.1–2.0 mm) and with citrate (20 mm). Colchicine (10^?6–10^?3m) inhibited enzyme activity and counteracted the effects of GTP and citrate. Sucrose density gradient centrifugation demonstrated that GTP and citrate preincubation promoted the formation of the polymeric, active enzyme, while colchicine engendered disassembly. Preincubation of the purified acetyl-CoA carboxylase at 4 °C caused inactivation and disassembly, which was countered by preincubation at 37 °C in the presence of GTP or citrate. These results suggest that GTP, like citrate, activates acetyl-CoA carboxylase by enhancing the conversion of the protomeric form of the enzyme to its more active, polymeric state.     

Metabolism of Monoterpenes: Acetylation of (-)-Menthol by a Soluble Enzyme Preparation from Peppermint (Mentha piperita) Leaves

The essential oil from mature leaves of flowering peppermint (Mentha piperita L.) contains up to 15% (—)-menthyl acetate, and leaf discs converted exogenous (—)-[G-³H]menthol into this ester in approximately 15% yield of the incorporated precursor. Leaf extracts catalyzed the acetyl coenzyme A-dependent acetylation of (—)-[G-³H]menthol and the product of this transacetylase reaction was identified by radiochromatographic techniques. Transacetylase activity was located mainly in the 100,000g supernatant fraction, and the preparation was partially purified by combination of Sephadex G-100 gel filtration and chromatography on O-diethylaminoethyl-cellulose. The transacetylase had a molecular weight of about 37,000 as judged by Sephadex G-150 gel filtration, and a pH optimum near 9. The apparent K_m and velocity for (—)-menthol were 0.3 mm and 16 nmol/hr· mg of protein, respectively. The saturation curve for acetyl coenzyme A was sigmoidal, showing apparent saturation near 0.1 mm. Dithioerythritol was required for maximum activity and stability of the enzyme, and the enzyme was inhibited by thiol directed reagents such as p-hydroxymercuribenzoate. Diisopropylfluorophosphate also inhibited transacylation suggesting the involvement of a serine residue in catalysis. The transacylase was highly specific for acetyl coenzyme A; propionyl coenzyme A and butyryl coenzyme A were not nearly as efficient as acyl donors (11% and 2%, respectively). However, the enzyme was much less selective with regard to the alcohol substrate, suggesting that the nature of the acetate ester synthesized in mint is more dependent on the type of alcohol available than on the specificity of the transacetylase. This is the first report on an enzyme involved in monoterpenol acetylation in plants. A very similar enzyme, catalyzing this key reaction in the metabolism of menthol, was also isolated from the flowers of peppermint.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and partial purification of dihydroxyacetone-phosphate acyltransferase from guinea pig liver

Dihydroxyacetone-phosphate:acyl coenzyme A acyltransferase (EC 2.3.1.42) was solubilized and partially purified from guinea pig liver crude peroxisomal fraction. The peroxisomal membrane was isolated after osmotic shock treatment and the bound dihydroxyacetone-phosphate acyltransferase was solubilized by treatment with a mixture of KCl-sodium cholate. The solubilized enzyme was partially purified by ammonium sulfate fractionation followed by Sepharose 6B gel filtration. The enzyme was purified 1200-fold relative to the guinea pig liver homogenate and 80- to 100-fold from the crude peroxisomal fraction, with an overall yield of 25–30% from peroxisomes. The partially purified enzyme was stimulated two- to fourfold by Asolectin (a soybean phospholipid preparation), and also by individual classes of phospholipid such as phosphatidylcholine and phosphatidylglycerol. The kinetic properties of the enzyme showed that in the absence of Asolectin there was a discontinuity in the reciprocal plot indicating two different apparent K_m values (0.1 and 0.5 mm) for dihydroxyacetone phosphate. The V_max was 333 nmol/min/mg protein. In the presence of Asolectin the reciprocal plot was linear, with a K_m = 0.1 mm and no change in V_max. The enzyme catalyzed both an exchange of acyl groups between dihydroxyacetone phosphate and palmitoyl dihydroxyacetone phosphate in the presence of CoA and the formation of palmitoyl [³H]coenzyme A from palmitoyl dihydroxyacetone phosphate and [³H]coenzyme A, indicating that the reaction is reversible. The partially purified enzyme preparation had negligible glycerol-3-phosphate acyltransferase (EC 2.3.1.15) activity.     

ATP Citrate Lyase from Germinating Castor Bean Endosperm: Localization and some Properties

ATP citrate lyase (EC 4.1.3.8) has been found in crude extracts from endosperm tissue of germinating castor bean and shows its maximum activity in 4- to 5-day-old seedlings. A strict requirement for coenzyme A and adenosine 5′-triphosphate was demonstrated. The pH optimum for the reaction is around 7.5. The unstable enzyme can be stabilized by freezing and addition of citrate and glycerol. (−)-Hydroxycitrate is a potent inhibitor. The molecular weight is about 400,000. The adenosine 5′-triphosphate citrate lyase is localized in the plastids, where it possibly plays a role in providing acetyl coenzyme A for lipid biosynthesis.     

Developmental changes in malonate-related enzymes of rat brain.

Mitochondria and high-speed supernatant were prepared from rat brain homogenates at 0–50 days of age. The development of malonyl-CoA synthetase, malonyl-CoA decarboxylase, coenzyme A-transferases and acetyl-CoA hydrolase was examined and compared to de novo fatty acid biosynthesis. The specific activity of malonyl-CoA synthetase rose steeply between 6 and 10 days, and this sudden increase coincided with peak specific activity of fatty acid synthetase. Similarly, malonate activation by coenzyme A-transfer from succinyl-CoA increased rapidly at the same time. Transfer of the coenzyme A moiety from acetoacetyl-CoA was only minimal during this period. Brain mitochondria had active malonyl-CoA decarboxylase which showed an almost linear increase of specific activity between 0 and 50 days. Acetyl-CoA resulting from malonyl-CoA decarboxylation underwent enzymatic hydrolysis to acetate and free coenzyme A. Only traces of acetoacetate were recovered. In mitochondria, acetyl-CoA hydrolase increased progressively whereas the cytosolic enzyme had high specific activity at birth which declined slowly during maturation.     

Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

Solubilization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat and chicken liver microsomes

A simple, efficient, freeze-thaw procedure for the solubilization of liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase has been developed. Microsomes of chicken or rat liver were prepared by homogenization in buffer containing 100 mm sucrose, 50 mm KCl, 40 mm KH₂PO₄, 30 mm EDTA, and 2 mm DTT, pH 7.2 (buffer A). The homogenate was centrifuged at 12,000g (15 min), and the microsomes were separated from the supernatant by centrifugation at 100,000g (60 min). The isolated microsomes were frozen, either by dry ice-acetone or by storage in a freezer at ?20°C. The frozen microsomes were permitted to thaw at room temperature, homogenized in buffer A, and centrifuged at 100,000g (60 min). The extraction was repeated and the combined supernatants contained 70 to 90% of the microsomal HMG-CoA reductase activity. The yield of enzyme activity by the freeze-thaw technique is equal to or greater than previously reported methodologies and is significantly easier to perform. This procedure is particularly suited to the preparation of large quantities of solubilized enzyme for isolation and characterization of HMG-CoA reductase. In addition, this method does not require the use of detergents, sonification, or other procedures which might partially inactivate or alter the molecular properties of the enzyme.     

Interaction of the fluorescent analogue stearoyl-(1,N6)-etheno coenzyme A with chicken liver acetyl coenzyme A carboxylase

The interaction of stearoyl-(1,N6)-etheno coenzyme A (stearoyl-epsilon-CoA) with acetyl coenzyme A carboxylase was investigated by using fluorescence spectroscopy. The fluorescence emission of stearoyl-epsilon-CoA was partially quenched by acetyl coenzyme A carboxylase. Analysis of the data for dissociation constant (KD) and the stoichiometry of the interaction (n) gave values of 5.06 nM and 1.2, respectively, at pH 7.6 in 50 mM Tris-HCl and 25 degrees C. The KD value is comparable to the inhibition constant (Ki) obtained previously by others for the inhibition of rat liver acetyl coenzyme A carboxylase by long chain fatty acyl-CoAs. Citrate (which is known to polymerize and thus activate carboxylase) caused a partial quenching of the protein fluorescence of carboxylase, presumably due to polymerization of the enzyme. The quenching of the stearoyl-epsilon-CoA fluorescence caused by carboxylase as well as the inhibition of carboxylase activity by stearoyl-epsilon-CoA was reversed by citrate, but only in the presence of 6-O-methylglucose polysaccharide which forms a stable complex with fatty acyl-CoA. This shows that the stearoyl-epsilon-CoA bound to the enzyme is displaced by citrate only in the presence of an acceptor of fatty acyl-CoA. These results support the reciprocal relationship of citrate and fatty acyl-CoA in the regulation of acetyl coenzyme A carboxylase.     

The requirement for a primer in the in vitro synthesis of polysaccharide by sweet-corn (1 → 4)-α-d-glucan syntrase

A mixture of (1 → 4)-α-d-glucan synthases was partially purified from sweet corn. The synthesis of polysaccharide from ADP-d-glucose by the enzyme preparation was dependent on added carbohydrate primer in solutions of low ionic strength, but displayed the phenomenon of being apparently primer-independent at high ionic strength in citrate buffer. This phenomenon was further investigated; treatment of the enzyme preparation with immobilized amylases led to the abolition of the apparently unprimed synthesis. The amylase-treated preparation then showed a normal dependence on (1 → 4)-α-d-glucan primer, branched primers being the most effective. The affinity of the enzyme for a branched primer appeared to be enhanced in the presence of citrate. The polysaccharide product of the unprimed reaction was glycogen-like, having an average chain-length of 14. These studies suggest that the phenomenon of unprimed synthesis in “high salt” is explicable in terms of an enhanced affinity of the enzyme for traces of primer in the enzyme preparation, and not to a “de novo” synthesis of polysaccharide, that occurs in the absence of a primer.     

Regulation of citrate synthase from blue-green bacteria by succinyl coenzyme A

Citrate synthase (EC 4.1.3.7) was prepared from nine species of blue-green bacteria. In every case the citrate synthase was of the large type otherwise found only in Gram-negative bacteria.In addition to inhibition by -oxoglutarate, the enzymes were all sensitive to inhibition by succinyl coenzyme A, acting competitively with respect to acetyl coenzyme A. Desensitization by potassium chloride and a sigmoidal dependence of inhibition on succinyl coenzyme A concentration suggested the possibility of an allosteric mechanism. Multiple-inhibition analysis using pairs of the competitive inhibitors succinyl coenzyme A, bromoacetyl coenzyme A and ATP confirmed the existence of a distinct site for succinyl coenzyme A.It is suggested that the specific sensitivity of bluegreen bacterial citrate synthases to succinyl coenzyme A, as well as to -oxoglutarate, is related to the particular metabolic role of the enzyme in these organisms. The absence of a complete energy-yielding citric acid cycle, resulting from the lack of -oxoglutarate dehydrogenase, confers a strictly biosynthetic role on citrate synthase, which initiates a branched pathway leading to the two end-products -oxoglutarate and succinyl coenzyme A. Inhibition of the enzyme by these compounds constitutes a plausible regulatory mechanism.     

Heat activation of rat liver acetyl-CoA carboxylase in vitro.

Acetyl-CoA carboxylase in rat liver homogenates was activated in vitro in a time- and temperature-dependent manner. The activity of acetyl-CoA carboxylase in rat liver preparations was determined in a 1-min assay to preclude the possibility of citrate activation of the enzyme during the assay period. Activation of the enzyme occurred more rapidly in liver preparations continuously maintained at ambient or greater temperatures than in homogenates of liver which had been chilled. High speed supernatant (105,000 X g, 60 min) did not heat-activate, and reconstitution of the heat-activatable 27,000 X g, 20-min, fraction by recombining the high speed pellet with the high speed supernatant only partially restored the heat activatability. Elution of the 105,000 X g supernatant from Sephadex G-25 resulted in an enzyme preparation which was heat-activatable. Addition of boiled 105,000 X g supernatant to the Sephadex G-25-treated enzyme again prevented heat activation. Dilution of the enzyme 5-fold did not prevent heat activation.     

Metabolism of trans-Aconitic Acid in Maize : I. PURIFICATION OF TWO MOLECULAR FORMS OF CITRATE DEHYDRASE

Trans-aconitate synthesis via citrate dehydrase was determined in crude extracts of maize (Zea mays L.) coleoptiles. Two molecular forms of this enzyme were purified by substrate-specific elution from DEAE-cellulose, ammonium sulfate precipitation, and gel filtration. Each molecular form migrates as a single band in isoelectric focusing. Gel filtration and sodium dodecyl sulfate electrophoresis provided evidence that one enzyme form is composed of four 80,000-dalton subunits while the other is composed of two 60,000-dalton subunits. There was no evidence of proteolytic conversion of the large to the small molecular weight form when the former was incubated with either the 15,000_g supernatant or with proteases. The data indicate that the two molecular forms of citrate dehydrase are isozymes.     

Amino Acid Catabolism and Malic Enzyme in Differentiating Dictyostelium discoideum

Amino acids produced from protein degradation are the major energy source for differentiation and aging in Dictyostelium discoideum. Considering the reactions involved in the conversion of amino acids from an average protein into tricarboxylic acid cycle intermediates, a route from a cycle intermediate (probably malate) to acetyl coenzyme A is required for the complete utilization of amino acids. Citrate was isolated from cells pulse-labeled with (14)C-labeled amino acids and was cleaved with citrate lyase. When cells were pulse-labeled with [U-(14)C]-glutamate the specific radioactivity of the acetate and oxaloacetate portions of citrate were consistent with the conclusion that one-third of the carbon flowing through the tricarboxylic acid cycle is removed for the synthesis of acetyl coenzyme A. The data were also consistent with the patterns of carbon flux required to maintain steady-state levels of cycle intermediates in cells catabolizing amino acids. It is suggested that the malic enzyme (EC 1.1.1.40) catalyzes the synthesis of acetyl coenzyme A from malate and is responsible for the observed citrate labeling pattern. In cell extracts the activity of this enzyme increased markedly with the onset of differentiation. The properties of partially purified (40-fold) malic enzyme isolated at culmination indicated that the enzyme was allosteric and was positively affected by aspartate and glutamate. Thus, amino acid production from protein degradation would stimulate a reaction essential for the efficient utilization of these amino acids for energy.     

Citrate lyase from Streptococcus diacetilactis

Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis. The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis The final enzyme preparation contained acetate: HS-citrate lyase ligase—an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase. This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase. Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme. Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity. The HS-form of citrate lyase from S. diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25° C. The enzyme did not acetylate the HS-lyases from R. gelatinosa and K. aerogenes. In contrast to the citrate lyases from R. gelatinosa and K. aerogenes the enzymes from S. diacetilactis and L. citrovorum showed onlya very weak reaction inactivation. It is assumed that this is due to the association of the acetylating enzymes with these lyases.     

Inactivation of citrate lyase from Rhodopseudomonas gelatinosa by a specific deacetylase and inhibition of this inactivation by L-(+1-glutamate.

A previously unrecognized enzyme, citrate lyase deacetylase, has been purified about 140-fold from cell extracts of Rhodopseudomonas gelatinosa. It catalyzed the conversion of enzymatically active acetyl-S-citrate lyase into the inactive HS-form and acetate. The enzyme exhibited an optimal rate of inactivation at pH 8.1. Because of the instability of acetyl-S-citrate lyase at acidic and alkaline pH values, all assays were carried out at pH 7.2, where the spontaneous hydrolysis of the acetyl-S-citrate lyase was negligible and deacetylase showed 70% of the activity at pH 8.1. The apparent Km value for citrate lyase was 10(-7) M at pH 7.2 and 30 C. The activity of the deacetylase was restricted to the citrate lyase from R. gelatinosa. The corresponding lyases from Enterobacter aerogenes (formerly Klebsiella aerogenes) and Streptococcus diacetilactis were not deacetylated; likewise, thioesters such as acetyl-S coenzyme A, acetoacetyl-S coenzyme A, and N-acetyl-S-acetyl-cysteamine were also not hydrolyzed. Citrate lyase deacetylase was present in very small amounts in cells of R. gelatinosa grown with acetate or succinate; it was induced by citrate along with the citrate lyase. L-(+)-Glutamate strongly inhibited the deacetylase. Fifty percent inhibition was obtained at a concentration of 1.4 X 10(-4) L-(+)-glutamate. D-(-)-Glutamate, alpha-ketoglutarate, L-alpha-hydroxyglutarate, L-(-)-proline, and other metabolites were less effective.     

Regulatory properties of citrate synthase from Rickettsia prowazekii

Citrate synthase [citrate (si)-synthase] (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (Rickettsia prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative molecular weight of approximately 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl coenzyme A saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP greater than ADP much greater than AMP). [beta,gamma-methylene]ATP, dATP, and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl coenzyme A was present in high concentration (greater than or equal to 50 microM). Neither NADH nor alpha-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eucaryotic and gram-positive procaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the first characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.     

Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6.

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).     

Reductive activation of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H.

When titanium(III) citrate was used as electron donor for the reduction of methyl coenzyme M by the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H, component A1 was no longer required. The simpler system thus obtained required components A2, A3, and C as well as catalytic amounts of ATP, vitamin B12, and the disulfide of 7-mercaptoheptanoylthreonine phosphate in addition to titanium(III) citrate. This three component enzyme system also could produce CH4 when stoichiometric amounts of 7-mercaptoheptanoylthreonine phosphate were used as a source of electrons under an H2 atmosphere. When 7-mercaptoheptanoylthreonine phosphate or H2 was used alone no CH4 was produced, indicating a dual requirement for reducing equivalents: one to activate the methylreductase system and the other to reduce methyl coenzyme M. This is the first evidence that the activation of methyl coenzyme M methylreductase is a reductive process.