Article

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

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Abstract

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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... The present work suggests that the pores produced in the membranes of Atriplex roots were probably in the range of 0.5 to 0.7 nm in radius since sugars, amino acids, and short chain organic acids could diffuse out relatively freely, whereas NAD was not able to reach the site of alcohol dehydrogenase. The pores induced in the membranes of microorganisms would appear to be larger since NAD passes freely (16, 19, 20). The slow outward diffusion of fluorescein from the cytoplasm observed under the fluorescence microscope is in accordance with the conclusion that the pores induced are not much larger than the Stokes radii of this molecule. ...
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Chapter
For many years now, the bacterium Acetobacter xylinum has served as a model organism for studying cellulose biogenesis, namely, the biosynthesis of linear β1,4-glucan chains and their crystallization into cellulose fibrils. In this chapter, we shall review developments in the study of the metabolic sequence leading from glucose to cellulose in this organism. We have not, however, attempted a complete survey of the literature; for a more detailed review of the field, the reader is referred to the review by Colvin (1972).
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The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in Acetobacter xylinum has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg(2+) ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (P(i)) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal pH for PEP synthesis was pH 8.2; for the reversal it was pH 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at pH 8.2 and 0.45 at pH 6.5. The apparent K(m) values of the components of the system in the forward reaction were: pyruvate, 0.2 mm; ATP, 0.4 mm; P(i), 0.8 mm; Mg(2+), 2.2 mm; and for the reverse reaction: PEP, 0.1 mm; AMP, 1.6 mum; PP, 0.067 mm; Mg(2+), 0.87 mm. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (K(i) = 0.2 mm). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by p-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in A. xylinum.
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A permeabilization method which allows the assay of several intracellular enzymes within the boundaries of the yeast cell wall is described. The kinetic properties of hexokinase and pyruvate kinase examined in the permeabilized cells, including the allosteric activation of the latter by fructose bisphosphate, are essentially the same as in cell-free extracts.
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Article
Glucose-grown cells of Acetobacter xylinum oxidized acetate only when the reaction mixture was supplemented with catalytic quantities of glucose or intermediates of the citrate cycle. Extracts, prepared by sonic treatment, catalyzed the formation of oxalacetate when incubated with phosphoenolpyruvate (PEP) and bicarbonate. Oxalacetate was not formed in the presence of pyruvate plus adenosine triphosphate. The ability to promote carboxylation of PEP was lower in succinate-grown cells than in glucose-grown cells. PEP carboxylase, partially purified from extracts by ammonium sulfate fractionation, catalyzed the stoichiometric formation of oxalacetate and inorganic phosphate from PEP and bicarbonate. The enzyme was not affected by acetyl-coenzyme A or inorganic phosphate. It was inhibited by adenosine diphosphate in a manner competitive with PEP (K(1) = 1.3 mm) and by dicarboxylic acids of the citrate cycle; of these, succinate was the most potent inhibitor. It is suggested that the physiological role of PEP carboxylase in A. xylinum is to affect the net formation of C(4) acids from C(3) precursors, which are essential for the maintainance of the citrate cycle during growth on glucose. The relationship of PEP carboxylase to other enzyme systems metabolizing PEP and oxalacetate in A. xylinum is discussed.
Article
Benziman, Moshe (The Hebrew University of Jerusalem, Jerusalem, Israel), and A. Abeliovitz. Metabolism of dicarboxylic acids in Acetobacter xylinum. J. Bacteriol. 87 270–277. 1964.—During the oxidation of fumarate or l-malate by whole cells or extracts of Acetobacter xylinum grown on succinate, a keto acid accumulated in the medium in considerable amounts. This acid was identified as oxaloacetic acid (OAA). No accumulation of OAA was observed when succinate served as substrate. These phenomena could be explained by the kinetics of malate, succinate, and OAA oxidation. OAA did not inhibit malate oxidation, even when present at high concentrations. When cells were incubated with OAA or fumarate in the presence of C¹⁴O2, only the beta-carboxyl of residual OAA was found to be labeled. Evidence was obtained indicating that nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) are not directly involved in malate oxidation by cell-free extracts. The results suggest that malate oxidation in A. xylinum is irreversible, and is catalyzed by an enzyme which is not NAD- or NADP-linked.