Amino acid sequence of the carboxy-terminal end of human erythrocyte glucose-6-phosphate dehydrogenase

Human erythrocyte glucose-6-phosphate dehydrogenase was purified to homogeneity by a simplified procedure, consisting of 2',5'-ADP-Sepharose affinity chromatography, followed by Sephadex G-100 gel filtration. The carboxy-terminal region of the protein was identified by carboxypeptidase digestion: the sequence -Lys-Leu-COOH was found instead of the reported -Gly-COOH, thus showing identity with the carboxy-terminal sequence of glucose-6-phosphate dehydrogenase from human leukocytes and platelets. In addition, the carboxyl-terminal peptide was isolated from a tryptic digest of the protein and sequenced. The sequence is: Trp-Val-Asp-Pro-His-Lys-Leu.     

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.     

Kinetic studies of Haemophilus influenzae 6-phosphogluconate dehydrogenase

Haemophilus influenzae 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate:NADP⁺ 2-oxidoreductase (decar☐ylating), EC 1.1.1.44) was purified 308-fold to electrophoretic homogeneity with a 16% recovery through a five-step procedure involving salt fractionation and hydrophobic and affinity chromatography. The purified enzyme was demonstrated to be a dimer of M_r 70 000, and to catalyze a sequential reaction process. The enzyme was NADP-specific and kinetic parameters for the oxidation of 6-phosphogluconate were determined for NADP and four structural analogs of NADP. Coenzyme-competitive inhibition by adenosine derivatives was significantly enhanced by the presence of a 2′-phosphoryl group consistent with the observed coenzyme specificity of the enzyme. The purified enzyme was effectively inhibited by 3-aminopyridine adenine dinucleotide phosphate, but at concentrations higher than that observed to inhibit growth of the organism. Rates of inactivation of the enzyme by N-ethylmaleimide were suggestive of sulfhydryl involvement in the reaction catalyzed.     

Amino acid sequence around a reactive cysteine of yeast alcohol dehydrogenase

The reaction of yeast alcohol dehydrogenase with iodoacetate produces a loss of 90% of the enzymatic activity when 2.2–2.4 equivalents of carboxymethyl groups are incorporated. After CNBr cleavage of the ¹⁴C-carboxymethylated enzyme the more radioactive fragment was maleylated and digested with trypsin. A tryptic peptide, containing about 70% of initial radioactivity, was purified and sequenced. The cysteine found to be more reactive with iodoacetate in our experimental conditions is different from that found in previous works. It also appears to differ from the cysteine residues found to be reactive towards other thiol reagents.     

Primate red cell enzymes: glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase

Glucose-6-phosphate dehydrogenase (E. C.: 1.1.1.49) phenotypes and 6-phosphogluconate dehydrogenase (E. C.: 1.1.1.44) phenotypes were determined by starch-gel electrophoresis of red cell hemolysates of Galago crassicaudatus subspp., Propithecus verreauxi, Lemur spp., Hapalemur griseus, and Macaca mulatta. A single glucose-6-phosphate dehydrogenase (G6PD) phenotype was found in each species. A single 6-phosphogluconate dehydrogenase (6PGD) phenotype was found in Lemur spp., Hapalemur griseus, and Galago crassicaudatus argentatus. In a group of six Propithecus verreauxi, three 6PGD phenotypes, PGD A, PGD AB, and PGD B, were found. Three phenotypes, PGD A, PGD AB, and PGD B, were found in 38 G. c. crassicaudatus. The three phenotypes in each species are apparently the products of two codominant autosomal alleles, PGD^A and PGD^B. The frequency of PGD^A in G. c. crassicaudatus is 0.263. A population of 260 free-ranging macaques displays a polymorphism at the 6PGD locus. Three phenotypes, PGD A, PGD AB, and PGD B, were found. These also appear to be controlled by two codominant autosomal alleles, PGD^A and PGD^B the frequency of PGD^A = 0.913. Additional analysis of three well-defined troops within the macaque population indicated that there are no significant differences between the troops or within the population at the 6PGD locus.     

Effectors of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver.

Summary The lower Vmax of 6PGDH with respect to G6PDH and its higher sensitivity to inhibition by NADPH, suggest the existence of an imbalance between the two dehydrogenases of the pentose phosphate pathway in rat liver. Possible modulators of these activities, particularly in relation with the inhibition by NADPH in physiological conditions, have been investigated. The results suggest that in both cases the inhibition by NADPH is strictly isosteric and that the relative affinities for the reduced and oxidized forms of the pyridine nucleotide are unaffected by glutathion, the intermediates of the pentose phosphate shunt or some divalent ions.Abbreviations G6PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - 6PGDH 6-phosphogluconate dehydrogenase (EC 1.1.1.44) On leave from the Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.     

Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver

The steady-state kinetics of the oxidative decarboxylation of 6-phosphogluconate catalysed by 6-phosphogluconate dehydrogenase from sheep liver in triethanolamine and phosphate buffers (pH 7.0) have been reinvestigated. In triethanolamine buffer the enzyme is inhibited by high NADP+ concentrations in the presence of low fixed concentrations of 6-phosphogluconate. Data are consistent with an asymmetric sequential mechanism in which NADP+ and 6-phosphogluconate bind randomly and product release is ordered. The pathway through the enzyme--6-phosphogluconate complex appears to be preferred in triethanolamine buffer. Pre-steady-state studies of the oxidative decarboxylation reaction at pH 6.0-8.0 show that hydride transfer is greater than 900 s-1. After the fast formation of NADPH in amounts equivalent to about half of the enzyme-active-centre concentration, the rate of NADPH formation is equal to the steady-state rate. Two possible interpretations are considered. Rapid fluorescence measurements of the displacement of NADPH from its complex with the enzyme at pH 6.0 and 7.0 indicate that the dissociation of NADPH is fast (greater than 800 s-1) and cannot be the rate-limiting step in oxidative decarboxylation. Coenzyme binding studies at equilibrium have been extended to include the determination of the dissociation constants for the binary complexes of enzyme with NADPH and NADP+ at pH 6.0-8.0 and the dissociation constant for NADPH in the ternary enzyme--6-phosphogluconate--NADPH complex in triethanolamine buffer, pH 7.0.     

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.     

Amino acid sequence of the phosphorylation site of isocitrate dehydrogenase fromEscherichia coli

InEscherichia coli, NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) may undergo a phosphorylation catalyzed by a cAMP-independent protein kinase, with a concomitant decrease in catalytic activity. In this report, we describe the purification and amino acid sequence of a³²P-labeled peptide obtained from in vivo³²P-labeled isocitrate dehydrogenase. The³²P-labeled peptide was isolated from a tryptic digest and found to contain seven amino acids, including a single serine residue. Following automated Edman degradation and reversephase high-pressure liquid chromatography of the phenylthiohydantoin-amino acids, the sequence of this peptide was established to be-Ser(P)-Leu-Asn-Val-Ala-Leu-Arg.