Concentration Of Acetyl Phosphate Research Articles

Acetate Metabolism and the Inhibition of Bacterial Growth by Acetate.

During aerobic growth on glucose, Escherichia coli excretes acetate, a mechanism called "overflow metabolism." At high concentrations, the secreted acetate inhibits growth. Several mechanisms have been proposed for explaining this phenomenon, but a thorough analysis is hampered by the diversity of experimental conditions and strains used in these studies. Here, we describe the construction of a set of isogenic strains that remove different parts of the metabolic network involved in acetate metabolism. Analysis of these strains reveals that (i) high concentrations of acetate in the medium inhibit growth without significantly perturbing central metabolism; (ii) growth inhibition persists even when acetate assimilation is completely blocked; and (iii) regulatory interactions mediated by acetyl-phosphate play a small but significant role in growth inhibition by acetate. The major contribution to growth inhibition by acetate may originate in systemic effects like the uncoupling effect of organic acids or the perturbation of the anion composition of the cell, as previously proposed. Our data suggest, however, that under the conditions considered here, the uncoupling effect plays only a limited role.IMPORTANCE High concentrations of organic acids such as acetate inhibit growth of Escherichia coli and other bacteria. This phenomenon is of interest for understanding bacterial physiology but is also of practical relevance. Growth inhibition by organic acids underlies food preservation and causes problems during high-density fermentation in biotechnology. What causes this phenomenon? Classical explanations invoke the uncoupling effect of acetate and the establishment of an anion imbalance. Here, we propose and investigate an alternative hypothesis: the perturbation of acetate metabolism due to the inflow of excess acetate. We find that this perturbation accounts for 20% of the growth-inhibitory effect through a modification of the acetyl phosphate concentration. Moreover, we argue that our observations are not expected based on uncoupling alone.

The role of acetyl-phosphate in the pathogenesis of Neisseria gonorrhoeae

Acetyl-phosphate (AcP), an intermediate from the phosphotransacetylase-acetate kinase (PTA-AK) pathway, has shown to be critical in pathogenic bacteria for the general metabolism and synthesis of virulence factors. Lysine acetylation is a post-translational modification (PTM) that occurs enzymatically and non-enzymatically by the addition of an acetyl residue from acetyl coenzyme A and AcP, respectively. Neisseria gonorrhoeae, the etiologic agent of gonorrhoea, has been shown to use AcP for lysine acetylation, however, the role that AcP has in the pathogenesis and how acetylation is regulated has not been discerned. The concentration of AcP was altered in N. gonorrhoeae MS11 by interrupting the genes involved in the PTA-AK pathway, pta and ackA, and the gene that encodes for a lysine deacetylase family protein, kdac. AcP concentrations were increased in ΔackA and decreased in Δpta resulting in modulation of lysine acetylation. Growth on glucose, lactate or pyruvate were investigated. In aerobic conditions, ΔackA mutant solely grew in glucose, while the Δpta mutant grew in glucose and lactate. In microaerophilic conditions, ΔackA and Δpta mutants solely grew in presence of glucose. The virulence of ΔackA and Δpta was tested by infecting larvae of Galleria mellonella. WT killed 50 % population (n=15) after 6 days and ΔackA after 24 h, however, Δpta after 6 days it only killed 10 %. Taken together, our results show AcP as an important metabolite for the metabolism and virulence of N. gonorrhoeae.

Cognate sensor kinase‐independent activation of Mycobacterium tuberculosis response regulator DevR (DosR) by acetyl phosphate: implications in anti‐mycobacterial drug design

The DevRS/DosT two-component system is essential for mycobacterial survival under hypoxia, a prevailing stress within granulomas. DevR (also known as DosR) is activated by an inducing stimulus, such as hypoxia, through conventional phosphorylation by its cognate sensor kinases, DevS (also known as DosS) and DosT. Here, we show that the DevR regulon is activated by acetyl phosphate under 'non-inducing' aerobic conditions when Mycobacterium tuberculosis devS and dosT double deletion strain is cultured on acetate. Overexpression of phosphotransacetylase caused a perturbation of the acetate kinase-phosphotransacetylase pathway, a decrease in the concentration of acetyl phosphate and dampened the aerobic induction response in acetate-grown bacteria. The operation of two pathways of DevR activation, one through sensor kinases and the other by acetyl phosphate, was established by an analysis of wild-type DevS and phosphorylation-defective DevSH395Q mutant strains under conditions partially mimicking a granulomatous-like environment of acetate and hypoxia. Our findings reveal that DevR can be phosphorylated in vivo by acetyl phosphate. Importantly, we demonstrate that acetyl phosphate-dependent phosphorylation can occur in the absence of DevR's cognate kinases. Based on our findings, we conclude that anti-mycobacterial therapy should be targeted to DevR itself and not to DevS/DosT kinases.

Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules

Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

The intracellular concentration of acetyl phosphate in Escherichia coli is sufficient for direct phosphorylation of two-component response regulators.

Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in Escherichia coli. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer.

Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria—the metabolism and potential for CO 2 recycling

Regulatory mechanism in PHB [poly-(hydroxybutyrate)] accumulation by cyanobacteria, especially by a thermophilic isolate, Synechococcus MA19 was reviewed in comparison with a genetically engineered strain. The strain, MA19 accumulates PHB under nitrogen starved and photoautotrophic conditions (MA19-N). Little PHB synthase activity was detected in crude extracts from the cells grown in nitrogen sufficient conditions (MA19+N). The activity was detected exclusively in membrane fractions from MA19+N. The change of the enzyme activity was insensitive to chloramphenicol, which suggests post-translational activation. In vitro, acetyl phosphate activated PHB synthase in membrane fractions from MA19+N, and the extent of activation depended on the concentration of acetyl phosphate. Phosphotransacetylase which catalyzes the conversion of acetyl-CoA to acetyl phosphate was detected in crude extracts from MA19-N but not in those from MA19+N. These results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C–N balance and intracellular acetyl-CoA concentration. On the contrary, in genetically-engineered cyanobacterium (transformant with PHB synthesizing genes from Ralstonia eutropha), it did not seem to be PHB synthase but acetyl-CoA flux that limits PHB synthesis. The closer association of PHB granules with thylakoid membranes in MA19 is suggested than that in the genetically-engineered cyanobacterium, which may reflect the difference of distribution of PHB synthase. Transposon-mutagenesis was used to acquire mutants of its altered PHB regulatory mechanism. PHA production by cyanobacteria was considered from the aspects of photobioreactors.

Regulation of RssB-dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator-controlled process.

Sigma(S) (RpoS) is a highly unstable global regulatory protein in Escherichia coli, whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock. As a consequence, these stresses result in the induction of sigma(S)-regulated stress-protective proteins. The two-component-type response regulator, RssB, is essential for the rapid proteolysis of sigma(S) and is probably involved in the transduction of some of these stress signals. Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo. Here, we demonstrate increased in vivo half-lives of sigma(S) and the RpoS742::LacZ hybrid protein (also a substrate for RssB-dependent proteolysis) in acetyl phosphate-free (pta-ackA) deletion mutants, even though no sensor kinase was eliminated. The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB. In vitro, efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp-58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed. Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild-type background. While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB-dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration.

Control of poly-beta-hydroxybutyrate synthase mediated by acetyl phosphate in cyanobacteria.

Poly-beta-hydroxybutyrate (PHB) synthesis in a cyanobacterium, Synechococcus sp. strain MA19, is controlled at the enzyme level and is dependent on the C/N balance in the culture medium. The control involves at least two enzymes. The first enzyme is PHB synthase. Little PHB synthase activity was detected in crude extracts from cells grown under nitrogen-sufficient conditions (MA19(+N)). The activity was detected exclusively in membrane fractions from nitrogen-deprived cells (MA19(-N)) under light but not dark conditions. The shift in the enzyme activity was insensitive to chloramphenicol, which suggests posttranslational activation. Acetyl phosphate activated PHB synthase in membrane fractions from MA19(+N). In vitro, the activation level of PHB synthase changed, depending on the concentration of acetyl phosphate. The second enzyme was phosphotransacetylase (EC 2.3.1.8), which catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate. The activity was detected in crude extracts from MA19(-N) but not in those from MA19(+N). The results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C/N balance and intracellular acetyl-CoA concentration. Acetyl phosphate probably acts as a signal of C/N balance affecting PHB metabolism in MA19.

Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli

We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation. Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature. Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis. To extend these observations, we measured the intracellular concentration of acetyl-CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates. Relative to increasing culture density, acetyl-CoA levels and expression from both the pta and ackA promoters decreased. Relative to increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased. In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter. We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl-CoA, the expression of ackA, and the activity of phosphotransacetylase.

Microenvironment of two different extrinsic fluorescence probes in Na+,K+-ATPase changes out of phase during sequential appearance of reaction intermediates.

Na+,K+-ATPase from pig kidney was sequentially modified with N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) at Cys-964 and fluorescein isothiocyanate (FITC) at Lys-501. The resulting preparation showed little Na+,K+-ATPase activity with retention of nearly 90% of phosphorylation capacity from acetyl phosphate. The addition of acetyl phosphate to the preparation induced phosphoenzyme formation with a sequential decrease in the fluorescence intensities in the presence of 2 M NaCl and 4 mM MgCl2; the BIPM fluorescence decreased with a simultaneous increase in the amount of phosphoenzyme; there was a significant delay in a decrease in the FITC fluorescence. The extent of the decrease in the BIPM fluorescence and the increase in the amount of phosphoenzyme both showed monophasic kinetics with a similar dependence on the concentration of acetyl phosphate (K0.5 = 4 mM), while that of FITC fluorescence showed a biphasic decrease (K 0.5 greater than 10 mM). The phosphoenzyme formed was insensitive to ADP but sensitive to acetate (K0.5 = 2 M). These data and those of others (Taniguchi, K., Suzuki, K., Kai, D., Matsuoka, I., Tomita, K., and Iida, S. (1984) J. Biol. Chem. 259, 15228-15233) showed that the extent of the decrease in the BIPM fluorescence reflects an increase in the amount of a precursor of E1P and E1P, irrespective of the FITC treatment. They also suggest the presence of at least two conformationally different E1Ps; one gave little and the other gave a large FITC fluorescence decrease.

Phosphatase activity of Na +/K +-ATPase. Enzyme conformation from ligands interactions and Rb occlusion experiments

The present work compares the effects of several ligands (phosphatase substrates, MgCl 2, RbCl and inorganic phosphate) and temperature on the phosphatase activity and the E 2(Rb) occuluded conformation of Na +/K +-ATPase. Cooling from 37°C to 20°C and 0°C (hydrolysis experiments) or from 20°C to 0°C (occlusion experiments) had the following consequences: (i) dramatically reduced the V max for p_ nitrophenyl phosphate and acetyl phosphate hydrolysis but it produced little or no changes in the K m for the subtrates; (ii) led to a 5-fold drop in the K m for the inorganic phosphate-induced di-occlusion of E 2(Rb); (iii) reduced the K 0.5 and curve sigmoidicity of the Rb-stimulated hydrolysis of p- nitrophenyl phosphate and acetyl phosphate and the Rb-promoted E 2(Rb) formation. At 20 °C, in the presence of 1 mM RbCl and no Mg 2+, acetyl phosphate did not affect E 2(Rb); with 3 mM MgCl 2, acetyl phosphate stimulated a release of Rb from E 2(Rb) both in the presence and absence of RbCl in the incubation mixture. As a function of acetyl phosphate concentration the K m for Rb release was indistinguishable from the K m found for stimulation of hydrolysis and enzyme phosphorylation under identical experimental conditions; in addition, the extrapolated di-occluded fraction corresponding to maximal dydrolysis was not different from 100%. These results indicate that although E 2(K) might be an intermediary in the phosphatase reaction, the most abundant enzyme conformation during phosphatase turnover is E 2 which has no K + occluded in it. The ligand interactions associated to phosphatase activity do not support an equivalence of this reaction with the dephosphorylation step in the Na ++ K + -dependent AT Phydrolysis; on the other hand, there are similarities with the reversible binding of inorgnic phosphate in the presence of Mg 2+ and K + ions.

Acetyl phosphate as a substrate for the calcium ATPase of sarcoplasmic reticulum.

Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt.

Acetylphosphate-induced Ca2+-Ca2+ exchange that is mediated by (Ca2+,Mg2+)-ATPase in sarcoplasmic reticulum vesicles.

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. 45Ca2+ efflux and influx were determined in the presence and absence of acetylphosphate. Phosphorylation of the membrane-bound (Ca2+,Mg2+)-ATPase by [32P]acetylphosphate was also determined. The rate of efflux with acetylphosphate was considerably higher than that without acetylphosphate. When the acetylphosphate concentration was greatly reduced by diluting the reaction mixture after the start of the reaction, the rate of the efflux decreased markedly. These results demonstrate the acceleration of 45Ca2+ efflux by acetylphosphate. This acetylphosphate-induced efflux required external Ca2+. The external Ca2+ concentration giving half-maximum activation of efflux was 3.8 microM. The Ca2+ concentration dependence of the efflux coincided with that of phosphorylation. When the acetylphosphate concentration was varied, the rate of acetylphosphate-induced efflux changed approximately in proportion to the phosphoenzyme concentration. These and other findings show that acetylphosphate-induced 45Ca2+ efflux represents Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme and further demonstrate the direct dissociation of Ca2+ from the Ca2+-bound phosphoenzyme to the external medium in Ca2+-Ca2+ exchange.

Evidence against an acyl-enzyme intermediate in the reaction catalyzed by clostridial phosphotransacetylase.

Clostridial phosphotransacetylase catalyzes acyl group transfer between coenzyme A (CoA) and inorganic phosphate and also the arsenolysis of acetyl-coenzyme A (AcCoA) to yield acetate and CoA-SH. The enzyme mobility on sodium dodecyl sulfate electrophoresis corresponds to a molecular weight of 70 000. Kinetics of both forward and reverse reactions are of the ternary type as previously reported and product inhibition data are consistent with a random binding scheme. One essential sulfhydryl group per 70 000 daltons was inactivated in a pseudo-first-order process by either N-ethylmaleimide or 5,5'-dithiobis (nitrobenzoic acid). Reduction of the rate of this inactivation by 50% in the presence of AcCoA or acetyl phosphate concentrations near their kinetic K values demonstrates binding of these acyl donors in simple enzyme-substrate complexes. Moreover, pulse-chase experiments show these binary complexes to be functional and also show that they do not dissociate rapidly compared with their rates of catalytic turnover. Incubation of the enzyme with 14C-labeled acyl donors failed to produce labeled protein after passage through Sephadex. This was true despite efforts to mimic "substrate synergism" with desulfo-CoA or to compensate for unfavorable equilibria by means of CoA traps. Very slow isotope exchange reactions of 32Pi into acetyl phosphate and [3H]CoA into AcCoA were at first observed. As in the cases of several other enzymes recently reexamined, these were shown on careful inspection to be artifacts of contamination by second substrates. Attempts to detect exchange reactions between acetyl phosphate and Pi, even in the presence of the CoA analogue, desulfo-CoA, were also unsuccessful. Therefore, no evidence for an acyl-enzyme could be detected. Furthermore, our data allow us to develop arguments which, we believe, indicate that an acyl-enzyme intermediate is extremely improbable in the reaction catalyzed by phosphotransacetylase.

Regulatory properties of acetokinase from Veillonella alcalescens.

Acetokinase from Veillonella alcalescens catalyzes the virtually irreversible synthesis of adenosine triphosphate from acetyl phosphate and adenosine diphosphate. Kinetic analysis revealed that the enzyme was activated by acetyl phosphate and inhibited by adenosine triphosphate. Velocity curves obtained with increasing amounts of adenosine diphosphate were of the Michaelis-Menten type (rectangular hyperbolas) under all conditions employed. However, velocity curves generated by varying the level of acetyl phosphate were sigmoidal, suggesting homotropic interactions for this substrate. Alteration of the pH of the reaction mixture from 7.4 to 10 or addition of a substrate analogue, propionyl phosphate, resulted in the loss of cooperativity and changed the values of the Hill coefficient from 3 to 1. Data obtained in experiments with propionyl phosphate suggested the existence of separate effector sites on the enzyme. The inhibition curves for adenosine triphosphate were also sigmoidal with Hill coefficients ranging between 1 and 3, depending on the concentration of acetyl phosphate. Sedimentation studies with the partially purified enzyme indicated a polymeric structure with a maximum molecular weight of about 90,000 daltons; a subunit of approximately 29,000 daltons was also detected.

Phosphotransacetylase of Escherichia coli B. activation by pyruvate and inhibition oby NADH and certain nucleotides

1. 1.|Phosphotransacetylase of Escherichia coli B has been found to be an allosteric enzyme which was activated by pyruvate and inhibited mainly by NADH. NAD + did not affect the enzyme activity. 2. 2.|ADP and ATP also inhibited the enzyme, But their inhibitory strengths were only one-quater and one-tenth of NADH, respectively. All these inhibitors affected the enzyme reaction, noncompetitively with CoA and sigmoidally with respect to the acetyl phosphate concentration. Pyruvate activated the enzyme by lowering K m for acetyl phosphate without a change in v max. A stimulatory site and an active site were distinct from each other. 3. 3.|Pyruvate repressed the inhibitory action of inhibitor and quite effectively activated the enzyme which had been inhibited by inhibitors, while it did not overcome the sigmoidicity of the reaction, indicating that inhibition and activation were based on separate mechanisms. 4. 4.|These inhibitors and activator were not consumed during the phosphotransacetylase acetylase reaction of E. coli B, in accordance with the result that phosphotransacetylase from Clostridium kluyveri was not affected by these effectors.

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.