2-keto-3-deoxy-6-phosphogluconate and 6-phosphogluconic-acid

Helicobacter pylori mutants deficient in 6-phosphogluconate dehydratase (6PGD) were constructed. Colonization densities were lower and minimum infectious doses were higher for mutant strains than for wild-type strains. In spite of better colonization, however, wild-type strains did not displace the mutant in cocolonization experiments. Loss of 6PGD diminishes the fitness of H. pylori in vivo, but the pathway is nonessential for colonization.

Topics: Animals; Colony Count, Microbial; Gastric Mucosa; Gluconates; Glycolysis; Helicobacter pylori; Hydro-Lyases; Mice; Mice, Inbred C57BL

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

Topics: Allosteric Regulation; Animals; Erythrocytes; Escherichia coli; Gluconates; Humans; Hydrogen-Ion Concentration; Kinetics; Liver; NADP; Phosphogluconate Dehydrogenase; Sheep; Trypanosoma brucei brucei

Evidence for the presence of the enzymes of the Entner-Doudoroff pathway in Helicobacter pylori was obtained using 1H and 31P nuclear magnetic resonance spectroscopy. Bacterial lysates generated 6-phosphogluconate and NADH or NADPH in incubations with glucose-6-phosphate and NAD+ or NADP+, indicating the presence of glucose-6-phosphate dehydrogenase activities. Formation of pyruvate was observed in time courses of incubations of bacterial lysates with 6-phosphogluconate as the only substrate, suggesting the presence of 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase activities. The existence of these enzymes and of triose phosphate isomerase was confirmed by observing the appearance of dihydroxyacetone phosphate in time courses of bacterial lysates incubated with 6-phosphogluconate. Aldolase activity was measured by the production of pyruvate and dihydroxyacetone phosphate in lysates incubated with 2-keto-3-deoxy-6-phosphogluconate as the sole substrate. Dehydrogenase, dehydratase and aldolase activities were observed in several bacterial strains including wild types from fresh isolates. Kinetic parameters were measured for the three activities. The cellular location of the enzymes was investigated by comparing the activities measured in the pellet and supernatant fractions obtained by centrifugation of lysate suspensions. The concentration of compounds causing 50% inhibition of enzyme activity was determined from dose-response curves. The data suggested the presence of two glucose-6-phosphate dehydrogenases linked to NAD+ and NADP+ activities. Using inhibitors differences between the H. pylori and mammalian KDPG aldolases were detected. The presence of these enzyme activities in H. pylori provided evidence for the existence of the Entner-Douderoff pathway in the bacterium.

Topics: Fructose-Bisphosphate Aldolase; Gluconates; Glucose; Glucose-6-Phosphate; Glucosephosphates; Helicobacter pylori; Hydro-Lyases; Magnetic Resonance Spectroscopy; NAD; NADP; Oxidation-Reduction; Oxidoreductases; Subcellular Fractions

Three major metabolites in the Entner-Doudoroff pathway, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, and pyruvate can be detected and quantified by HPLC with pulsed amperometric detection. Resolution is achieved by ion-exchange chromatography at alkaline pH with isocratic elution in 5 to 10 min. Detection limits are in the subnanomolar range, and detector response is linear over 3-4 orders of magnitude. This method can be employed for the assay of the enzymes of the pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14), eliminating the need for coupling enzymes as in the previously employed spectrophotometric assays. The lag in pyruvate production seen in the coupled enzyme spectrophotometric assay for 6-phosphogluconate dehydratase is absent in the HPLC/pulsed amperometric detection assay. This lag represents an artifact of a slow tautomerism of 2-keto-3-deoxy-6-phosphogluconate which must precede its utilization by the coupling enzyme, 2-keto-3-deoxy-6-phosphogluconate aldolase. Kinetic data on the approach to equilibrium of 2-keto-3-deoxy-6-phosphogluconate aldolase-catalyzed interconversion of 2-keto-3-deoxy-6-phosphogluconate, pyruvate, and glyceral-dehyde-3-phosphate can be also accurately quantified by HPLC with pulsed amperometric detection.

Topics: Aldehyde-Lyases; Azotobacter vinelandii; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrochemistry; Gluconates; Glucose; Hydro-Lyases; Hydrogen-Ion Concentration; Kinetics; Pyruvates; Sensitivity and Specificity

Mortlock, R. P. (U.S. Army Chemical Corps, Frederick, Md.). Gluconate metabolism of Pasteurella pestis. J. Bacteriol. 84:53-59. 1962.-During a study of gluconate metabolism by a virulent strain of Pasteurella pestis, evidence was obtained for the presence of gluconokinase, 6-phosphogluconate dehydrogenase, transketolase, and 2-keto-3-deoxy-6-phosphogluconate dehydrase in cell-free extracts. A study of the products of 6-phosphogluconate degradation by extracts indicated that 6-phosphogluconate was metabolized by both the 6-phosphogluconate dehydrogenase-transketolase pathway and the Entner-Doudoroff pathway. No evidence could be obtained for the presence of an active glucose-6-phosphate dehydrogenase.

Topics: Carbohydrate Metabolism; Gluconates; Glucose; Oxidoreductases; Phosphogluconate Dehydrogenase; Yersinia pestis