The role of 6-phosphogluconate dehydrogenase in Rhizobium.

A nicotinamide adenine dinucleotide (NAD) linked 6-phosphogluconate (6-PG)dehydrogenase has been detected in Rhizobium. The enzyme activity is similar in both slow- and fast-growing rhizobia. The nicotinamide adenine dinucleotide phosphate (NADP) dependent 6-PG dehydrogenase was detected only in the fast growers and was more than twice as active as the NAD-linked enzyme. Partial characterization of the products of 6-PG oxidation in Rhizobium suggests that the NADP-linked enzyme is the decarboxylating enzyme of the pentose phosphate (PP) pathway (EC 1.1.1.44) whereas a phosphorylated six-carbon compound, containing ketonic group(s), is the product of the oxidation catalyzed by the NAD-linked enzyme.     

Fluorescence studies of coenzyme binding to 6-phosphogluconate dehydrogenase.

The fluorescence quantum yield of NADPH is enhanced in its complex with 6-phospho-gluconate dehydrogenase, and a further enhancement in the presence of excess 6-phospho-gluconate shows that an abortive ternary complex is formed. There is marked energy transfer from aromatic residues in the enzyme to NADPH in the complexes, as indicated by an excitation maximum at 280 nm in the fluorescence excitation spectrum of the complex. The coenzyme fluorescence enhancement has been used to determine the dissociation constant for NADPH in the binary and ternary complexes, and the stoichiometry of the complexes, from the results of fluorescence titrations. A new method of analysis of fluorescence titration data is described. The results show that each subunit of the dimeric enzyme binds NADPH independently and with the same affinity. The dissociation constant for the enzyme-coenzyme complex, in phosphate buffer, pH 7.0, is 5.7 μm; the dissociation constant for NADPH in the ternary complex with 6-phosphogluconate is 7.0 μm.     

Interactions of inhibitors at the coenzyme binding site of 6-phosphogluconate dehydrogenase.

Nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) was purified 200-fold from chicken erythrocyte nuclei. An important feature of the purification procedure is the preliminary preparation of chromatin and extraction of the enzyme from insoluble chromatin into 0.3 m NaCl. Active enzyme in a partially purified preparation has an isoelectric point of 5.5 and a molecular weight of approximately 300,000. The most highly purified enzyme migrates as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a broad pH optimum from 6–9 and is most active at 55 °C. The activation energy for the enzyme-catalyzed reaction is 8.0 kcal/mol. The purified enzyme is able to use either nicotinate mononucleotide or nicotinamide mononucleotide as a substrate. The isolation procedure is applicable to partial purification of enzyme activity from erythrocytes of closely related birds, including pheasant, goose, and turkey. Immunochemical studies of the enzyme are reported in an accompanying article.     

Use of multiple isotope effects to study the mechanism of 6-phosphogluconate dehydrogenase

The multiple isotope effect method of Hermes et al. [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114] has been used to study the mechanism of the oxidative decarboxylation catalyzed by 6-phosphogluconate dehydrogenase from yeast. 13C kinetic isotope effects of 1.0096 and 1.0081 with unlabeled or 3-deuterated 6-phosphogluconate, plus a 13C equilibrium isotope effect of 0.996 and a deuterium isotope effect on V/K of 1.54, show that the chemical reaction after the substrates have bound is stepwise, with hydride transfer preceding decarboxylation. The kinetic mechanism of substrate addition is random at pH 8, since the deuterium isotope effect is the same when either NADP or 6-phosphogluconate or 6-phosphogluconate-3-d is varied at fixed saturating levels of the other substrate. Deuterium isotope effects on V and V/K decrease toward unity at high pH at the same time that V and V/K are decreasing, suggesting that proton removal from the 3-hydroxyl may precede dehydrogenation. Comparison of the tritium effect of 2.05 with the other measured isotope effects gives limits of 3-4 on the intrinsic deuterium and of 1.01-1.05 for the intrinsic 13C isotope effect for C-C bond breakage in the forward direction and suggests that reverse hydride transfer is 1-4 times faster than decarboxylation.     

Alkylation of 6-phosphogluconate dehydrogenase from Candida utilis with coenzyme analogues.

The mechanism of the inactivation of 6-phosphogluconate dehydrogenase from Candida utilis with two coenzyme analogues can be differentiated on the basis of kinetic studies and of the properties of the inactivated enzyme. 3-Chloroacetylpyridine--adenine dinucleotide phosphate is clearly an affinity label and 3-choloroacetylpyridine--adenine dinucleotide a second-order reagent. For 3-chloroacetylpyridine--adenine dinucleotide phosphate, there is a loss of one thiol per subunit at complete inactivation whereas for 3-chloroacetylpyridine--adenine dinucleotide 2.7 thiol groups are lost. The fluorescence of the protein is quenched after alkylation by 3-chloroacetylpyridine--adenine dinucleotide phosphate and there is no quenching after the inactivation with 3-chloroacetylpyridine--adenine dinucleotide.     

The 6-phosphogluconate dehydrogenase reaction in Escherichia coli.

This study is an attempt to relate in vivo use of the 6-phosphogluconate dehydrogenase reaction in Escherichia coli with the characteristics of the enzyme determined in vitro. 1) The enzyme was obtained pure by affinity chromatography and kinetically characterized; as already known, ATP and fructose-1,6-P2 were inhibitors. 2) A series of isogenic strains were made in which in vivo use of thereaction might differ, e.g. a wild type strain versus a mutant lacking 6-phosphogluconate dehydrase, as grown on gluconate; a phosphoglucose isomerase mutant grown on glucose or glycerol. 3) The in vivo rate of use of the 6-phosphogluconate dehydrogenase reaction was determined from measurements of growth rate and yield and from the specific activity of alanine after growth in 1-14C-labeled substrates. 4) The intracellular concentrations of 6-phosphogluconate, NADP+, fructose-1,6-P2, and ATP were measured for the strains in growth on several carbon sources. 5) The metabolite concentrations were used for assay of the enzyme in vitro. The results allow one to calculate how fast the reaction would function in vivo if ATP and fructose-1,6-P2 were its important effectors and if the in vitro assay conditions apply in vivo. The predicted in vivo rates ranged down to as low as one-tenth of the actual rates, and, accordingly, one cannot yet draw firm conclusions about how the reaction is actually controlled in vivo.     

The stabilization by a coenzyme analog of a conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

The reaction of NADP⁺ with periodate yields a coenzyme analog that can be bound to the NADP⁺ binding site of 6-phosphogluconate dehydrogenase from Candida utilis. This coenzyme analog can be irreversibly bound to the enzyme by reduction with sodium borohydride. The binding of one molecule of inhibitor to only one of the two subunits of the enzyme causes the inactivation of this subunit but does not alter the catalytic activity of the other subunit. Thus the two subunits do not have apparent catalytic interactions. When the reaction between the enzyme and the coenzyme analog is carried out in the presence of the substrate, the covalent modification of only one subunit causes the inactivation of both subunits. In this case the two subunits show an extreme negative cooperativity. It is suggested that the binding of the substrate induces in the enzyme molecule a conformational change that is stabilized by the irreversible binding of the coenzyme analog.     

The singly-wound parallel beta barrel: a proposed structure for 2-keto-3-deoxy-6-phosphogluconate aldolase.

Two short local reconnections in the backbone chain tracing of 2-keto-3-deoxy-6-phosphogluconate aldolase suffice to make it an 8-stranded parallel β barrel whose size, shape, topology, and connection handedness match those of triose phosphate isomerase and of the first domain of pyruvate kinase. It is proposed that this singly-wound parallel β barrel is in fact the tertiary structure of the aldolase subunit.     

Importance in catalysis of the 6-phosphate-binding site of 6-phosphogluconate in sheep liver 6-phosphogluconate dehydrogenase

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.