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Factors afecting the activity of pyruvate kinase of Acetobacter xylinum.

ABSTRACT: 1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg(2+) ions for activity. It was not activated by K(+) or NH(4) (+) ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis-Menten kinetics. 5. The enzyme was inhibited by ATP (K(i)0.9x10(-3)m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg(2+) ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.

SUBMITTER: Benziman M

PROVIDER: S-EPMC1187766 | biostudies-other | 1969 May

REPOSITORIES: biostudies-other

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Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

Project description:The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.

| S-EPMC1172560 | biostudies-other

Studies on recombinant Acetobacter xylinum alpha-phosphoglucomutase.

Project description:The phosphoglucomutase (PGM) from Acetobacter xylinum, which had been cloned and expressed in Escherichia coli, has been studied. After expression, the enzyme was purified from the E. coli in a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography. The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 microM BSA. The isoelectric point for A. xylinum PGM was 4.8 and the molar absorbance was 3.9 x 10(4) M-1.cm-1. The enzyme was reasonably heat-stable below 50 degrees C and was stable throughout the pH 5.5-7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+. The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A. xylinum. Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+. The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 microM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2 x 10(4) M-1.s-1. The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.

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| S-EPMC1166239 | biostudies-other

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| S-EPMC54906 | biostudies-other

The structure of novel C35 pentacyclic terpenes from Acetobacter xylinum.

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Project description:Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of beta-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, and cdg3. Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.

| S-EPMC107450 | biostudies-literature

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Project description:A second cellulose synthase gene (acsAII) coding for a 175-kDa polypeptide that is similar in size and sequence to the acsAB gene product has been identified in Acetobacter xylinum AY201. Evidence for the presence of this gene was obtained during analysis of A. xylinum mutants in which the acsAB gene was disrupted (I.M. Saxena, K. Kudlicka, K. Okuda, and R.M. Brown, Jr., J. Bacteriol. 176:5735-5752, 1994). Although these mutants produced no detectable cellulose, they exhibited significant cellulose synthase activity in vitro. The acsAII gene was isolated by using an acsAB gene fragment as a probe. The acsAII gene coded for cellulose synthase activity as determined from sequence analysis and study of mutants in which this gene was disrupted. A mutant in which only the acsAII gene was disrupted showed no significant differences in either the in vivo cellulose production or the in vitro cellulose synthase activity compared with wild-type cells. Mutants in which both the acsAII and acsAB genes were disrupted produced no cellulose in vivo and exhibited negligible cellulose synthase activity in vitro, thus confirming that the cellulose synthase activity observed in the acsAB mutants was coded by the acsAII gene. These results establish the presence of an additional gene for cellulose synthase expressed in cells of A. xylinum, yet this gene is not required for cellulose production when cells are grown under laboratory conditions.

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Project description:1. Whole cells of Acetobacter xylinum were found to contain a quinone of the ubiquinone (coenzyme Q) group. The quinone was isolated from the cells and crystallized. It was identified by its physical, chemical and spectroscopic properties as a ubiquinone with 10 isoprene units (ubiquinone-10). No naphthaquinone was detected in the cells. 2. Cell-free extracts prepared by means of a French pressure cell were separated into three fractions by differential centrifugation. The ubiquinone was located predominantly in the particulate fraction sedimenting at 33000g, which also contained most of the NADH oxidase and malate oxidase activities. The concentration of ubiquinone-10 in extracts was similar to that of the flavoproteins and about three times the concentration of the individual cytochromes. 3. Aerobic incubations of crude extracts with either NADH or malate resulted in reduction of the endogenous ubiquinone-10 to steady-state concentrations of 55 and 40% of the total quinone respectively. In the presence of cyanide more than 95% of the endogenous ubiquinone-10 was reduced by either NADH or malate. 4. The initial rate of reduction of endogenous ubiquinone-10 by malate and the rate of ubiquinol oxidation, in A. xylinum extracts, were found to be compatible with the overall rate of malate oxidation with oxygen. 5. The effects of various respiratory inhibitors on the oxidation-reduction reactions of the endogenous quinone indicate that its position on the respiratory chain is between the malate flavoprotein dehydrogenase and the cytochrome chain.

| S-EPMC1198809 | biostudies-other

AG-348 enhances pyruvate kinase activity in red blood cells from patients with pyruvate kinase deficiency.

Project description:Pyruvate kinase (PK) deficiency is a rare genetic disease that causes chronic hemolytic anemia. There are currently no targeted therapies for PK deficiency. Here, we describe the identification and characterization of AG-348, an allosteric activator of PK that is currently in clinical trials for the treatment of PK deficiency. We demonstrate that AG-348 can increase the activity of wild-type and mutant PK enzymes in biochemical assays and in patient red blood cells treated ex vivo. These data illustrate the potential for AG-348 to restore the glycolytic pathway activity in patients with PK deficiency and ultimately lead to clinical benefit.

| S-EPMC5609468 | biostudies-literature

Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene.

Project description:The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported. The sequence data indicated that the gene product consists of 284 amino acids. This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.

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