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

Swissa, M; Benziman, Moshe

doi:10.1042/bj1530173

Cited by 17 publications

(12 citation statements)

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“…The citrate synthase of A. europaeus was inhibited by ATP and was not affected by NADH and NADPH. This property is consistent with the findings in A. xylinum [12] and Methylobacillus flagellatum [9]. Due to the purification to electrophoretic homogeneity of our protein, the specific activity of the A. europaeus citrate synthase was 7‐times higher than in A. xylinum .…”

Section: Discussionsupporting

confidence: 91%

“…europaeus citrate synthase (20 μM) is different from that of A. xylinum (9 μM) [12]. The K m value for acetyl‐CoA of A. europaeus citrate synthase (51 μM) is within the lowest range of the corresponding values from Gram‐negative bacteria and is different from that of A. xylinum (18 μM) [12]. The kinetic properties of the A. europaeus citrate synthase differ from those of Gram‐negative bacteria.…”

Section: Discussionmentioning

confidence: 84%

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Purification and properties of citrate synthase from Acetobacter europaeus

Sievers

Stöckli

Teuber

2006

FEMS Microbiology Letters

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Citrate synthase (EC 4.1.3.7) was purified from the acidophilic bacterium Acetobacter europaeus to electrophoretic homogeneity. The specific activity was 228 units/mg of protein during the exponential ethanol-oxidation growth phase. The enzyme has a molecular mass of 280 kDa and is a hexamer with a subunit size of 46 kDa. The apparent K(m) values were 20 microM for oxaloacetate and 51 microM for acetyl-CoA. Unlike citrate synthase from other Gram-negative bacteria, the activity of the enzyme was inhibited by ATP, slightly enhanced by ADP and not effected by NADH. Acetate caused activation of the enzyme. The pH optimum on the citrate synthase activity in vitro was 8.1. The amino-terminal amino acid sequence of the purified enzyme was ENGKSATISLNGKDVALPVL.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 84%

Purification and properties of citrate synthase from Acetobacter europaeus

Sievers

Stöckli

Teuber

2006

FEMS Microbiology Letters

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“…Citrate synthase activity was assayed with 5,5'dithiobis-(2-nitrobenzoic acid) (Swissa & Benziman, 1973, 1976Srere et al, 1963). Glycerokinase activity was determined by t-he formation from _4C]glycerol ofglycerol 3-phosphate, which was adsorbed on discs of DEAE-cellulose filter paper and then its radioactivity counted, and dihydroxyacetone kinase activity by measuring the oxidation of NADH in a reaction coupled with glycerol 3-phosphate dehydrogenase (Weinhouse &Benziman, 1975;Torner, 1975).…”

Section: Methodsmentioning

confidence: 99%

“…The citrate synthase isolated and purified from Acetobacter xylinum extracts was shown (Swissa & Benziman, 1913, 1976 to be inhibited by ATP and to be insensitive to NADH, thus differing from the enzyme isolated from other Gram-negative bacteria. It has been suggested that the flux through the citric acid cycle in A. xylinum is regulated -by modulation of citrate synthase activity in response to the energy state of the cells (Swissa & Benziman, 1973, 1976.…”

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confidence: 99%

Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro

Swissa

Weinhouse

Benziman

1976

Biochemical Journal

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The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.

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“…Previous studies of B. subtilis CS activity were based on crude extracts (5, 9), now known to contain a mixture of CS-I, the product of citA, and CS-II. Several CSs from gram-negative bacteria have been purified and their kinetic and biochemical properties have been studied in detail (14,16,17,21,22), but only one CS enzyme from a gram-positive bacterium (Bacillus megaterium) has been studied previously in its homogeneously purified form (18). Bacillus sp.…”

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confidence: 99%

Characterization of the major citrate synthase of Bacillus subtilis

Jin

Sonenshein

1996

J Bacteriol

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The major citrate synthase of Bacillus subtilis (CS-II) was purified to near homogeneity and shown to correspond to the product of the citZ gene. Accumulation of CS-II during exponential growth and stationary phases paralleled expression of the citZ gene. The physical and kinetic properties of CS-II were similar to those of citrate synthase enzymes from Bacillus megaterium and from eukaryotic cells but differed from those of citrate synthases from many gram-negative bacteria.Citrate synthase (CS), the first enzyme of the Krebs cycle, is required for synthesis of glutamate from glycolytic intermediates and is, therefore, a critical enzyme in cellular biosynthesis (23). Moreover, the role of the Krebs cycle in production of ATP and reducing power makes this enzyme a major contributor to cellular energetics. In Bacillus subtilis, CS is needed for both growth (in the absence of a source of glutamate) and spore formation (5-7).B. subtilis has two distinct, homologous CS genes, citA and citZ, expressed during growth and early stationary phase (7,8). A null mutation in citA has little effect on growth, CS enzyme activity, or sporulation, but an in-frame deletion mutation in citZ causes partial glutamate auxotrophy, a Ͼ90% loss of CS enzyme activity, and a significant defect in sporulation. A citA citZ double mutant is an absolute glutamate auxotroph, has undetectable CS activity, and sporulates more poorly than does either a citA or citZ single mutant. These results, along with comparative measurements of citA and citZ mRNA levels, have indicated that citZ encodes the major CS of B. subtilis (8).Here we report the biochemical and kinetic properties of purified CS-II, the citZ gene product. Previous studies of B. subtilis CS activity were based on crude extracts (5, 9), now known to contain a mixture of CS-I, the product of citA, and CS-II. Several CSs from gram-negative bacteria have been purified and their kinetic and biochemical properties have been studied in detail (14,16,17,21,22), but only one CS enzyme from a gram-positive bacterium (Bacillus megaterium) has been studied previously in its homogeneously purified form (18). Bacillus sp. strain C4 CS has been studied after partial purification (19).To purify B. subtilis CS-II without contamination by CS-I, we used a citA null mutant strain, SJB9, a derivative of JH642 (7). Purification followed the method of Robinson et al. (18) with minor modifications (see the legend to Fig. 1 and Table 1). B. subtilis CS activity in crude extracts and in pure preparations was found to be rapidly lost at 4ЊC unless protected by 20% glycerol. In the presence of 20% glycerol and 100 mM KCl, the purified B. subtilis enzyme retained its activity for more than 6 months at 4ЊC and for about 2 years at Ϫ20ЊC.The N-terminal amino acid sequence (MTATRGLEGV VATTSSVSSII) and the sequence of an internal tryptic peptide (MLTEIGEVEN) were identical to those predicted from the DNA sequence of the citZ gene (7). These results very strongly suggest that citZ encodes CS-II. Molecular mass and olig...

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

Cited by 17 publications

References 28 publications

Purification and properties of citrate synthase from Acetobacter europaeus

Purification and properties of citrate synthase from Acetobacter europaeus

Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro

Characterization of the major citrate synthase of Bacillus subtilis

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