P alpha-chiral phosphorothioate analogues of bis(5''-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.

Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae. The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S. No dithiophosphate analogue was formed. Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively. The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate. The adenylate was accepted by corresponding thioanalogues of ATP. In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60. The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E. coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S. cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S. For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively. Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.     

Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli

Acetate kinase from Salmonella typhimurium and Escherichia coli was purified to electrophoretic homogeneity. The amino acid compositions of both proteins were similar, and the apparent molecular weights were the same, about 40,000 for the putative monomers. The native proteins gave higher molecular weights, suggesting that the enzymes may be oligomers, perhaps with two polypeptide subunits. Steady-state kinetic studies were performed with the enzymes isolated from both organisms and the kinetic constants were determined. The Km values were 0.07 and 7 mM for ATP and acetate, respectively. In contrast to earlier studies using less pure preparations, the homogeneous enzymes from both strains were active only with acetate but not with propionate or butyrate. The enzyme activity was cold-labile, and the length of reactivation time in the presence of Mg X ATP and acetate was dependent on protein concentration, suggesting that the monomer may not be catalytically active. The enzyme was phosphorylated with [gamma-32P]ATP and the phosphoprotein was isolated. Phosphoacetate kinase was capable of transferring the phosphate group to either ADP or acetate. The accompanying paper (Fox, D. K., Meadow, N. D., and Roseman, S. (1986) J. Biol. Chem. 261, 13498-13503) shows that the phosphoryl group of phosphoacetate kinase can also be reversibly transferred to Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system.     

The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma-bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor.     

Comparative bioenergetics of sulfate reduction in Desulfovibrio and Desulfotomaculum spp.

Extracts of Desulfotomaculum nigrificans, Desulfotomaculum orientis, and Desulfotomaculum ruminis exhibit low levels of inorganic pyrophosphatase but were found to have high levels of pyrophosphate:acetate phosphotransferase. Conversely, extracts of Desulfovibrio gigas, Desulfovibrio vulgaris, and Desulfovibrio desulfuricans Norway 4 were shown to have high levels of inorganic pyrophosphatase but negligible amounts of pyrophosphate:acetate phosphotransferase. Both enzymes are reductant activated and appear to have an analogous function in removing pyrophosphate formed during the activation of sulfate. Conservation of the bond energy of pyrophosphate in Desulfotomaculum eliminates the necessity for invoking electron-transfer-coupled phosphorylation to account for the growth of these bacteria on lactate plus sulfate. Relative growth yields of Desulfovibrio vulgaris and Desulfotomaculum orientis on lactate plus sulfate indicate that the latter does not carry out significant electron-transfer-coupled phosphorylation in this mode of growth.     

A reinvestigation of dynein ATPase kinetics and the inhibitory action of vanadate

The kinetic properties of sea urchin flagellar dynein ATPase have been reinvestigated using a continuous assay which regenerates ATP and contains P1,P5-di(adenosine-5')pentaphosphate, a potent adenylate kinase inhibitor. Earlier studies (Shimizu, T. (1981) Biochemistry 20, 4347-4354) revealed complex, highly cooperative kinetics with respect to MgATP2- concentration in the absence of this inhibitor. With Ap5A, the kinetics are characteristic of classical Michaelis-Menten enzymes. Isolated 21 S and 14S enzyme forms were also examined, and their kinetic parameters are presented Vanadate inhibition patterns in the presence of P1,P5-di(adenosine-5')pentaphosphate lose their nonlinear character, and we observe linear noncompetitive inhibition of the "mixed" type.     

Reaction mechanism and structural model of ADP-forming Acetyl-CoA synthetase from the hyperthermophilic archaeon Pyrococcus furiosus: evidence for a second active site histidine residue

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P(i) acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the beta-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (alpha(2)beta(2)) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His-71beta. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:     

The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.     

Sulfate-Dependent Interspecies H(2) Transfer between Methanosarcina barkeri and Desulfovibrio vulgaris during Coculture Metabolism of Acetate or Methanol

We compared the metabolism of methanol and acetate when Methanosarcina barkeri was grown in the presence and absence of Desulfovibrio vulgaris. The sulfate reducer was not able to utilize methanol or acetate as the electron donor for energy metabolism in pure culture, but was able to grow in coculture. Pure cultures of M. barkeri produced up to 10 mumol of H(2) per liter in the culture headspace during growth on acetate or methanol. In coculture with D. vulgaris, the gaseous H(2) concentration was 相似文献   

Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris hildenborough using gas chromatography-mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry

Flux distribution in central metabolic pathways of Desulfovibrio vulgaris Hildenborough was examined using 13C tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that >80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.     

The EutQ and EutP proteins are novel acetate kinases involved in ethanolamine catabolism: physiological implications for the function of the ethanolamine metabolosome in Salmonella enterica

Salmonella enterica catabolizes ethanolamine inside a compartment known as the metabolosome. The ethanolamine utilization (eut) operon of this bacterium encodes all functions needed for the assembly and function of this structure. To date, the roles of EutQ and EutP were not known. Herein we show that both proteins have acetate kinase activity and that EutQ is required during anoxic growth of S. enterica on ethanolamine and tetrathionate. EutP and EutQ‐dependent ATP synthesis occurred when enzymes were incubated with ADP, Mg(II) ions and acetyl‐phosphate. EutQ and EutP also synthesized acetyl‐phosphate from ATP and acetate. Although EutP had acetate kinase activity, ΔeutP strains lacked discernible phenotypes under the conditions where ΔeutQ strains displayed clear phenotypes. The kinetic parameters indicate that EutP is a faster enzyme than EutQ. Our evidence supports the conclusion that EutQ and EutP represent novel classes of acetate kinases. We propose that EutQ is necessary to drive flux through the pathway under physiological conditions, preventing a buildup of acetaldehyde. We also suggest that ATP generated by these enzymes may be used as a substrate for EutT, the ATP‐dependent corrinoid adenosyltransferase and for the EutA ethanolamine ammonia‐lyase reactivase.     

Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii.

In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.     

Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a K(m) of 4.26x10(-5)m for RS-mevalonate and 1.54x10(-3)m for ATP. The preparation from green leaves had a K(m) of 4.55x10(-5)m for RS-mevalonate and 1.75x10(-3)m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.     

Pyruvate dehydrogenase and the path of lactate degradation in Desulfovibrio vulgaris Miyazaki F

Pyruvate dehydrogenase from Desulfovibrio vulgaris Miyazaki F was partially purified from the soluble fraction of the bacterial sonicate, and characterized. The enzyme catalyzes oxidative decarboxylation of pyruvate to produce acetyl-CoA, in contrast to statements in current review articles in which acetyl phosphate is indicated to be a direct decomposition product of pyruvate in sulfate-reducing bacteria. The established reaction stoichiometry is: pyruvate + CoA + FMN----acetyl-CoA + CO2 + FMNH2. The Km values are 2.9 mM for pyruvate, 32 microM for CoA and 6.7 mumol for FMN. Participation of thiamine diphosphate in the enzymic process was not proven. 2-Oxobutyrate, but not 2-oxoglutarate, can substitute for pyruvate. The three flavin compounds, FMN, FAD, and flavodoxin, as well as clostridial ferredoxin, serve as electron carriers for the enzyme. Thus the enzyme is a kind of pyruvate synthase [EC 1.2.7.1], but acts in the direction of pyruvate degradation in the growing cells. The rate of cytochrome C3 reduction is extremely low, but in the presence of flavodoxin as an electron mediator, the reduction rate of cytochrome C3 becomes faster than the reduction rate of flavodoxin alone. It seems that the physiological electron acceptor for this enzyme is flavodoxin, which might be complexed with cytochrome C3 to produce a very efficient electron transfer system in the cell. The soluble fraction of D. vulgaris cells has been proved to contain, in addition to the pyruvate dehydrogenase, lactate dehydrogenase (Ogata, M., Arihara, K., & Yagi, T. (1981) J. Biochem. 89, 1423-1431), phosphate acetyltransferase and acetate kinase, i.e., all the enzymes necessary to convert lactate to acetate, producing ATP by substrate level phosphorylation.     

Cloning and expression of the enolase gene from Desulfovibrio vulgaris (Miyazaki F)

The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 2.1-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene (eno) and part of the methylenetetrahydrofolate dehydrogenase gene (folD). The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids. An expression system for eno under control of the T7 promoter was constructed in E. coli. The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of D-(+)-2-phosphoglyceric acid (2-PGA). The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied. The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D. vulgaris was grown on lactate.     

The tricarboxylic and acid pathway in Desulfovibrio.

Strains of two species of Desulfovibrio were examined for enzymes of the tricarboxylic acid cycle and related pathways. Pyruvate carboxylase (EC6.4.1.1) is present, and alpha-ketoglutarate is formed via the tricarboxylic acids. Glutamate, but not succinyl-CoA, arises from alpha-ketoglutarate. A pathway exists from pyruvate by malic enzyme (EC 1.1.1.39) activity to malate, then fumarate and succinate, again with no evidence of succinyl-CoA formation. The enzymes concerned with metabolism of these dicarboxylic acids show greater activity in the strains that can grow by fumarate dismutation. Glutamate (or glutamine), alpha-ketoglutarate, and yeast extract repress the enzymes that metabolize the tricarboxylic acids. There appears to be no glyoxylate cycle in Desulfovibrio vulgaris or D. desulfuricans.     

Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase,phosphofructokinase and pyruvate kinase

Summary The activities of hexokinase (ATP:hexose-6-phosphate transferase, E.C. 2.7.1.1), phosphofructokinase (ATP: fructose-6-phosphate 1-phosphotransferase, E. C. 2.7.1.11) and pyruvate kinase (ATP: pyruvate transferase, E.C. 2.7.1.40), and their kinetic behaviour in two morphological forms of Trypanosoma cruzi (epimastigotes and metacyclic trypomastigotes) have been studied. The kinetic responses of the three enzymes to their respective substrates were normalized to hyperbolic forms on a velocity versus substrate concentration plots. Hexokinase and phosphofructokinase showed a higher activity in epimastigotes than in metacyclics, whereas pyruvate kinase had similar activity in both forms of the parasite. The specific activity of hexokinase from epimastigotes was 102.00 mUnits/mg of protein and the apparent Km value for glucose was 35.4 M. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3 M. The kinetic parameters (specific activity and Km for fructose 6-phosphate) of phosphofructokinase for epimastigotes were 42.60 mUnits/mg and 0.31 mM and for metacyclics 13.97 mUnits/mg and 0.16 mM, respectively. On the contrary, pyruvate kinase in both forms of T. cruzi did not show significant differences in its kinetic parameters. The specific activity in epimastigotes was 37.00 mUnits/mg and the Km for phosphoenolpyruvate was 0.47 mM, whereas in metacyclics these values were 42.94 mUnits/mg and 0.46 mM, respectively. The results presented in this work, clearly demonstrate a quantitative change in the glycolytic pathway of both culture forms of T. cruzi.Abbreviations NNN Novy-Nicolle-McNeal medium - Eagle's MEM Eagle's Minimal Essential Medium with Earle's salts - IFCS heat Inactivated Fetal Calf Serum 56°C, 30 min) - Tris tris(hydroxymethyl) aminomethane - EDTA Ethylenediaminetetraacetic Acid     

一株硫酸盐还原菌的分离鉴定和系统发育分析

从处理硫酸盐废水的厌氧折流板反应器中分离得到一株硫酸盐还原菌D11, 该菌株革兰氏反应阴性, 无芽孢, 菌体杆状稍有弯曲, 宽度在0.6 μm~0.8 μm, 长度在1.8 μm~3.3 μm之间, 有极生单鞭毛, 能运动, 接触酶阳性, 氧化酶阴性。菌株生长的pH范围介于6.0~8.0之间, 最适pH为7.0, 生长温度范围为25°C~37°C, 最适温度为30°C。能够以葡萄糖、蔗糖、乙酸、乳酸、乙醇和丙二醇为唯一碳源生长, 不能利用丙三醇、丁醇、琥珀酸和苹果酸。菌株DNA的G+C含量为62.7 mo     

Structure of the metal-nucleotide complex in the acetate kinase reaction. A study with gamma-32P-labeled phosphorothioate analogs of ATP

The synthesis of the gamma-32P-labeled diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and the Sp isomer of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) by a modification of the Glynn and Chappell method (Glynn, I. M., and Chappell, J. T., (1964) Biochem. J. 90, 147-149) is described. These analogs were tested as substrates for acetate kinase in the presence of several divalent metal ions. Both isomers of ATP alpha S are substrates in the presence of Mg2+, Mn2+, Co2+, Zn2+, and Cd2+, the Sp isomer being preferred by a factor of between 4.8 (Mg2+) and 52.5 (Cd2+). Only the Rp isomer of ATP beta S is a substrate in the presence of Mg2+, and the Sp isomer becomes a better substrate in the presence of Mn2+, Co2+, and Zn2+; both isomers are equally good substrates in the presence of Cd2+. The change in specificity upon replacing Mg2+ by Cd2+ is greater than 1800 at beta-phosphorus and 10 at alpha phosphorus. These results provide a basis for proposing that the lambda screw sense configuration of the beta, gamma-bidentate MgATP complex is the substrate for acetate kinase. In the reverse reaction, both Sp and Rp isomers of ADP alpha S are substrates in the presence of all metal ions tested, the Sp isomer preferred by a factor between 12.3 (Mg2+) and 45.5 (Cd2+). In the presence of Mg2+, Mn2+, and Co2+, only the Rp isomer of ATP beta S is synthesized from prochiral ADP beta S, while a mixture of Rp and Sp isomers is synthesized in the presence of Zn2+ and Cd2+. These results are analogous to those for the forward reaction and suggest that the Mg.ADP complex which binds as a substrate in the reverse reaction, and is released as a product in the forward reaction, is the beta-monodentate. The classification of acetate kinase as an enzyme having a type I mechanism (Dunaway-Mariano, D. and Cleland, W. W. (1980) Biochemistry 19, 1506-1515) for kinases, is discussed.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H(2) on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H(2) and CO(2) for methanogenesis, degraded lactate, with the production of acetate and CH(4). When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H(2)-CO(2) and acetate for methanogenesis, lactate was stoichiometrically degraded to CH(4) and presumably to CO(2). During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH(4). Later, acetate was degraded to CH(4) and presumably to CO(2). In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H(2) was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H(2), but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH(4) from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H(2)-CO(2) as the methanogenic substrate produced CH(4) from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H(2) produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.