Acetate Phosphotransferase Research Articles

Role of energetic coenzyme pools in the production of l-carnitine by Escherichia coli

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into l(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into l(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD + ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD + pool indicated that it is correlated with the biotransformation process at the NAD + regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD + ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).

From ‘reactive C 2 units’ to acetyl coenzyme A: a long trail with an acetyl phosphate detour

Discovery of CoA and acetyl CoA diminished interest in acetyl phosphate, but it has a role in many bacterial fermentations. It is formed from acetate and ATP by the action of acetate kinase (EC 2.7.2.1) and by three other enzymatic reactions. Acetate kinase, when named systematically, is ATP:acetate phosphotransferase – hence, in the direction of ATP synthesis this enzyme uses acetyl phosphate in Lipmann’s second splitting mode16xDevelopment of the acetylation problem, a personal account. Lipmann, F. Science. 1954; 120: 855–865Crossref | PubMedSee all References, 17xSee all References as a phosphate donor. This transfer is a major route for ATP formation in some bacteria.Acetate kinase, together with adenosine kinase and adenylate kinase, has been used in an ingenious, large-scale synthesis of ATP from ADP with acetyl phosphate as phosphate donor28xLarge-scale enzyme-catalysed synthesis of ATP from adenosine and acetyl phosphate. Regeneration of ATP from ADP. Baughn, R.L. et al. J. Am. Chem. Soc. 1978; 100: 304–306CrossrefSee all References28. The sum of three separate reactions is as follows: adenosine + 3 acetyl phosphate = ATP + 3 acetate. The acetyl phosphate, required in large amounts, was obtained (as diammonium salt) by the keten reaction19xA new synthesis of acetyl dihydrogen phosphate. Bentley, R. J. Am. Chem. Soc. 1948; 70: 2183–2185Crossref | PubMedSee all References19.After the discoveries of CoA and acetyl CoA, progress was rapid. Acetyl CoA was found to play a crucial role in the tricarboxylic acid cycle by reaction with oxaloacetate to form citrate. Another carrier for acyl groups, acyl carrier protein (ACP) was discovered; it contains the same pantetheine moiety as CoA. Both CoA and ACP activate a variety of acids, for example, in fatty acid anabolism. In this process, it was established that the extending C2 units derive from malonyl CoA; this further CoA derivative was formed from acetyl CoA by the biotin-dependent acetyl CoA carboxylase (EC 6.4.1.2). The trail from ‘activated acetate’ to acetyl CoA provided a truly bountiful harvest of information. Moreover, four Nobel Prizes had involved work with CoA in one way or another!

Kinetics and Mechanism of Acetate Kinase From Bacillus Stearothermophilus.

The initial rates of ATP regeneration catalyzed by acetate kinase (ATP: acetate phosphotransferase; EC 2.7.2.1) from Bacillus stearothermophilus were measured under a wide range of MgADP–, acetyl phosphate, MgATP2– and acetate concentrations. The experimental results showed that the enzyme exhibited positive co-operativity for MgATP2– and no co-operativity for MgADP–, acetyl phosphate and acetate. Furthermore, the initial rates of the forward reaction, in which MgATP2– and acetate were produced from MgADP– and acetyl phosphate, were inhibited by MgATP2– and acetate, and those of the reverse reaction were inhibited by MgADP– and acetyl phosphate. The initial rate data were correlated well by using the rate equation derived based on the assumption that the reaction obeys the general reaction scheme based on the Random Bi Bi mechanism, and on the previous experimental result that the enzyme behaves like a dimeric enzyme, even though it is a tetrameric enzyme.

Immobilization of acetate kinase and phosphotransacetylase on derivatized glass beads

The enzymes acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1) and phosphotransacetylase (acetyl coenzyme A: orthophosphate acetyltransferase, EC 2.3.1.8) were separately immobilized onto controlled pore glass CPG and silica beads (pore size 50 nm). Different coupling techniques were screened and immobilized enzymes were subjected to storage stability tests. The selected method, the CPG γ-aminopropyl glutaraldehyde succinate dihydrazide, was further optimized to improve the activity of the enzyme-loaded glass beads.

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min −1 and 0.051 min −1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.

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.

Inactivation of Escherichia coli acetate kinase by [formula omitted] protection by substrates and products

Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N- ethylmaleimide at micromolar concentrations. However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained. When incubated with 7.2 μM N- ethylmaleimide , acetate kinase was inactivated with a rate constant of 0.063 min −1. Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl 2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation. While both acetate and acetyl phosphate did not affected the protection rendered by AMP, the presence of acetly phosphate altered ADP protection. However, both substrates prevented ATP from protecting the enzyme. These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.

Acetate kinase: a triple-displacement enzyme.

Facts relating to the mechanism of phosphoryl transfer by acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) are reviewed. They point to the existence of at least one experimentally established phosphoenzyme (E-P) intermediate on the reaction pathway. Sterically, the phosphoryl transfer occurs with a net inversion of the configuration of the phosphorus atom. These facts are best in accord with a triple-displacement mode of action for acetate kinase, with two E-P intermediates and three steric inversions on phosphorus. It follows that a second E-P for acetate kinase must exist.

Requirements of acetyl phosphate for the binding protein-dependent transport systems in Escherichia coli.

In Escherichia coli, acetyl phosphate can be formed from acetyl-CoA via the phosphotransacetylase (phosphate acetyltransferase; acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) reaction and from acetate (plus ATP) via the acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) reaction. By restricting acetyl phosphate formation to the phosphotransacetylase reaction alone, through the use of metabolic inhibitors, we were able to show that, with pyruvate as a source of energy, mutants defective in phosphotransacetylase are unable to transport glutamine, histidine, and methionine. However, with the same energy source, mutants defective in acetate kinase are normal in the transport of these amino acids. The inability of the phosphotransacetylase mutants to transport is due to their presumed inability to form acetyl phosphate, because pyruvate is found to be metabolized to acetyl-CoA in these mutants. Thus acetyl phosphate has been implicated in active transport. Evidence is also presented that neither the protonmotive force nor the ecf gene product is required for the shock-sensitive transport systems.

The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli.

Mutants of Escherichia coli K12 have been isolated that grow on media containing pyruvate of proline as sole carbon sources despite the presence of 10 or 50 mM-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP: acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA: orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta- and Ack- mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate: CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.

La germination de la spore de clostridum tyrobutyricum: II. Démonstration de l'intervention de l'acétokinase et de la phosphotransacétylase par l'étude de leurs propriétés

Summary The properties of an acetokinase AK (ATP: acetate phosphotransferase, E.C. 2.7.2.1) and a phosphotransacetylase PTA (acetyl-CoA: orthophosphate acetyl transferase, E.C. 2.3.1.8) isolated from vegetative cells of C. tyrobutyricum have been studied in order to investigate if they are involved in spore germination. These enzymes are similar to those from other microrganisms especially. E. coli for AK and C. kluyveri for PTA. AK has a molecular weight of 68,000 daltons. It phosphorylates acetate and at a smaller rate propionate. The K m values are 100 mM for acetate, 135 mM for propionate, 3 mM for ATP, 5 mM for ADP and 3 mM for acetyl-P. The equilibrium constant of the AK reaction, which is markedly in favor of ATP and acetate, has been estimated to be 0.005 and the mechanism has been found to be «Ping-Pong. AK has an absolute requirement for Mg 2+ or Mn 2+ ; it is strongly inhibited by Mg + and p-CMB and activated by reducing agents. The phosphorylation of acetate by AK is inhibited competitively by the following compounds: fluoroacetate > butyrate ≥ thioglycolate > lactate > glycolate and propionate; formate, glycine, iodoacetate and acetamide have no effect. PTA has a molecular weight of 58,000 daltons. The reaction catalyzed by PTA is reversible but the rate of acetylation of CoA is much greater than the hydrolysis of acetyl-CoA. The K m values are 1.1 mM for acetyl-P and 0.03 mM for CoA. K + and particularly NH 4 + activates PTA, but Na + , Li + , Cs + and Ca 2+ inhibit it. Na + is a competitive inhibitor with respect to NH 4 + . Enzyme activity is also dependent on the anions, the relative order of effectiveness is: Cl − > CH 3 -COO − > SO 4 2− > PO 4 3− . NADH, ADP, ATP and pyruvate display no significant effect on PTA but a non-competitive inhibition with acetyl-P was observed with 3 and 2.3-PGA and a mixed inhibition with 2-PGA. The comparison of cell and spores AK and PTA on the basis of their pH optima and their interaction with substrates and inhibitors did not show significant differences. It is concluded that AK and PTA are involved in the initiation of spore germination in C. tyrobutyricum because of the complete agreement between several properties of these enzymes, especially the effect of inhibitors, and those of germination. An hypothesis on the role of acetyl-CoA is suggested.

Purification and properties of acetate kinase from Clostridium thermoaceticum.

Acetate kinase (ATP: acetate phosphotransferase EC 2.7.2.1) has been purified from Clostridium thermoaceticum. The enzyme of a specific activity of 282 μmoles min-1 mg-1 appeared homogeneous as judged from Sephadex chromatography and sedimentation velocity. Polyacrylamide gel electrophoretic patterns at pH 9.0 and 9.5 showed heterogeneity. Velocity curves obtained with varying amount of acetate were of the Michaelis-Menten type with an apparent K m of 0.135 M. With varying amounts of ATP sigmoidal kinetic was observed (S0.5=1.64 mM), suggesting cooperative binding of this substrate. The enzyme had only moderate thermal stability with a temperature optimum of about 60°C and exhibited a broken line in an Arrhenius graph. From gel filtration a molecular weight of about 60 000 daltons was estimated for the enzyme. The S20w value was 6.0 S.

Phosphorylated Acetate Kinase: ITS ISOLATION AND REACTIVITY

Abstract Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) can be phosphorylated with ATP or with acetyl phosphate. The degree of phosphorylation increases with the relative concentration of phosphorylating agent. The isolated phosphoenzyme reacts quantitatively with ADP to form ATP, and with acetate to form acetyl phosphate in 70 to 80% yield. The pH stability profile of the phosphoenzyme at 37° has a U shape characteristic of acyl phosphates. Neutral hydroxylamine at 26° reacts rapidly (10 min) with the phosphoenzyme to effect the quantitative release of orthophosphate. These properties intimate that the phosphoryl group of the phosphoenzyme is linked covalently to a carboxyl function of the enzyme.

Evaluation of the phosphoryl-enzyme intermediate concept in the acetate kinase and hexokinase reactions from kinetic studies

The kinetic mechanism of action of Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1) was found to be Ping Pong. This conclusion was alluded to from initial rate experiments and from competitive and product inhibition experiments. The findings are consistent with chemical evidence providing the basis for the participation of a phosphoryl-enzyme intermediate in the reaction. A discussion is presented in which it is shown that an analogous covalent intermediate is most probably not involved in the reaction catalyzed by yeast hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7.1.1). Finally, a reinvestigation of the use of isotope competition for segregating kinetic mechanisms of multisubstrate enzymic reactions indicates that the approach is invalid.

Exchange Reactions Catalyzed by Acetate Kinase

Abstract Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) catalyzes an ADP-ATP and an acetate-acetyl phosphate exchange. The rate of the nucleotide exchange is unaffected by the presence of acetate. The specific rate of the ADP-ATP exchange is 200 times greater than that of the acetate-acetylphosphate exchange. The exchanges and the over-all reaction have pH rate profiles which are separate and distinct from one another. Mercuric ion inhibits to the same degree the over-all reaction and the acetate-acetyl phosphate exchange but is without effect on the ADP-ATP exchange. Catalysis of the two exchanges accords with the mediation of a phosphoenzyme in the action of acetate kinase.

A Phosphoenzyme Intermediary in Acetate Kinase Action

Abstract Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) has been phosphorylated with 32P, 14C-acetyl phosphate. Only phosphorus was transferred to the enzyme. Phosphoenzyme, prepared with acetyl phosphate, can synthesize ATP from ADP and acetyl phosphate from acetate.

Control of pyruvate phosphoroclastic activity in extracts of Clostridium pasteurianum by ADP and acetyl phosphate

1. 1. The ATP requirement for the pyruvate phosphoroclastic reaction has been investigates in extracts of Clostridium pasteurianum, particularly with regard to control of the system. 2. 2. S-Adenosylmethione is not the active component in the ATP requirement in pyruvate metabolism in C. pasteurianum although it is known to be in Escherichia coli and Streptococcus faecalis. 3. 3. AMP and ADP can replace ATP, since enzymes in the extract establish an equilibrium mixture of the three. The active species has been shown to b ADP. 4. 4. Acetyl phosphate is a product inhibitor of the reaction. ADP, through its involvement in the acetate kinase (ATP: acetate phosphotransferase, EC 2.7.4.3) reaction, appears to exert its effect by influencing acetyl phosphate concentration. 5. 5. Arsenate removes the requirement for adenosine phosphate by preventing the accumulation of acetyl phosphate. 6. 6. Pyruvate consumption by extracts of ammonia-grown cells is not stimulated by the addition of ATP. Addition of arsenate, or ATP + ATPase (ATP phosphohydrolase, EC 3.6.1.4), stimulates pyruvate breakdown by preventing the accumulation of acetyl phosphate which otherwise occurs. 7. 7. Increasing ADP concentration counters some of the inhibitory effect of acetyl phosphate, indicating a direct control by ADP in addition to its involvement in acetyl phosphate breakdown. 8. 8. A scheme for the control of pyruvate phosphoroclastic activity in C. pasteurianum is proposed: acetyl phosphate acts as a coarse control and ADP as a fine regulator.

The adenosine triphosphate requirement for nitrogen fixation in cell-free extracts of Clostridium pasteurianum

1.The requirements for the decomposition of acetyl phosphate in extracts of Clostridium pasteurianum have been shown to be the same as those for N2 fixation with H2 as the electron donor—H2, ATP, ferredoxin and Mg2+. The decomposition of acetyl phosphate (ATP) under H2 is not caused by an activation of enzymes by H2, nor by an ATP-dependent uptake of substrate amounts of H2.2.Removal of ATP from extracts by charcoal treatment allowed N2 fixation from pyruvate to proceed appreciably when fixation from H2 was almost completely inhibited. Addition of increasing amounts of ATP to extracts freed of ATP by Sephadex gel-filtration showed that a much lower concentration of ATP-supported N2 fixation from pyruvate than from H2. One site of ATP action was thus localized between H2 and reduced ferredoxin.3.A requirement for ATP in pyruvate metabolism has been established and may explain the ATP requirement for N2 fixation from pyruvate. It appears doubtful that ATP is required at the site of N2 reduction.4.H2 acted as a competitor of N2 fixation when H2 was the electron donor, but acetyl phosphate (ATP) consumption increased with increasing pH2. When N2 fixation was completely inhibited by either CO or N2O, ATP consumption was only slightly decreased. Separate enzymes apparently function in ATP consumption and N2 reduction.5.Mg2+ is required for ATP consumption as well as for ATP generation from acetyl phosphate by acetokinase (ATP: acetate phosphotransferase, EC 2.7.2.1).6.A scheme is presented for the action of ATP in N2 fixation; an activated form of reduced ferredoxin produced enzymically with ATP is considered to function in N2 fixation.

Acetataktivierung in grünalgen: II. Bie bildung von acetylphosphat in Stichococcus bacillaris und anderen arter der chlorococcales

1. 1. Resting cells of Stichococcus bacillaris rapidly metabolise acetate. In contrast to former experiments with Euglena gracilis there was no lag phase in O 3 -uptake. Immediately after addition of acetate to respiring cells O 2 -uptake rose to maximum level. Only about 50% of the added acetate is oxidises, the remainder is assimilated 2. 2. Although in crude cell-free preparations acethydroxamic acid formation occurred also after CoA had been removed, there was a small stimulative effect upon adding CoA to the mixture. In reaction mixtures, however, which had been heated for 20 min to 55°, CoA had no influence on the formation of hydroxamic acid. 3. 3. In short time experiments authentic acetyl phosphate will be metabolised by crude extracts only if ADP as a phosphate acceptor is present. Exact stoichimometric relations exist between the disappearance of acetyl phosphate and ADP and the synthesis of ATP. 4. 3. All experiments performed provide strong evidence, that in Stichococcus bacillaris and some other species of Chlorococcales acetate activation is initiated by acetyl phosphate formation. The enzyme responsible for this reaction, acetate-kinase (ATP: acetate phosphotransferase, EC 2.7.2.1), has been purified. The requirements for the reaction were studied, and the Michaelis constants determined.

An acetate-activating system from rumen microorganisms

Abstract An acetate-activating system was isolated from rumen microorganisms. Ammonium sulfate fractions of an aqueous acetone powder extract were used as a source of enzyme for this study. These extracts were shown to possess the capability of activating acetate to its CoA ester via an acetokinase-phosphotransacetylase activation scheme. In this study, the acetokinase (ATP:acetate phosphotransferase, EC 2.7.2.1) portion of this system was shown to be stimulated by addition of K+, Rb+ or NH4+ to the assay medium. Optimum concentration of these ions were 0.02–0.04 M and the relative order of effectiveness was K+ > Rb+ > NH4+. The reaction was also found to be inhibited by Na+ or Li+, but niether of these ions inhibited competitively. Acetokinase also displayed a requirement for divalent cations which could be satisfied by either Mg2+ or Mn2+ but not by Ba2+ or Ca2+. When the concentrations of both K+ and Mg2+ were varied, it was observed that the optimum K+ level could be decreased by as much as four-fold by increasing the Mg2+ concentration. Studies in which the ATP to Mg2+ ratio was held constant indicated that maximum activity occurred when the ATP/Mg2+ ratio was between 0.33 and 0.67.