H.Richard Levy

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: Interaction of the enzyme with coenzymes and coenzyme analogs 1

Abstract Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD+ or NADP+ as coenzyme. Kinetic studies showed that NAD+ and NADP+ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057) . In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP+ and 50% for NAD+. The dissociation constant for NADP+, determined from fluorescence quenching measurements, is 3 μ m , which is similar to the previously determined Km of 5.7 μ m and Ki of 5 μ m . The dissociation constant for NAD+ is 2.5 m m , which is 24 times larger than the previously determined Km of 0.106 m m . Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD+ but not for NADP+. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD+ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD+; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μ m ; the previously determined Ki is 37.6 μ m . The dissociation constant for NAD′ is 2.8 m m , in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP+-competitive and NAD+-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD+ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.

On the molecular weight of human glucose 6-phosphate dehydrogenase

Abstract Glucose 6-phosphate dehydrogenase was isolated by a rapid procedure from lactating rat mammary glands, bovine erythrocytes and several human tissues, including erythrocytes. Molecular weights of the subunits and active forms of all these enzymes were found to be approximately the same and not to differ significantly from the molecular weights of other mammalian glucose 6-phosphate dehydrogenases. The difference between the earlier reported values for the molecular weight of human erythrocyte glucose 6-phosphate dehydrogenase and the present results is suggested to arise from possible modification of the enzyme during earlier, lengthy isolation procedures.

On the absence of cysteine in glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides

Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has a subunit molecular weight of 55,000, determined by SDS gel electrophoresis. The amino acid composition of the enzyme is reported. An unusual feature which has not been reported for any other dehydrogenase, is the complete absence of cystine or cysteine. This establishes the fact that cysteine is not an obligatory participant in the mechanism of action of NAD(P)-linked dehydrogenases.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: Revised kinetic mechanism and kinetics of ATP inhibition

The kinetic mechanisms of the NAD- and NADP-linked reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides were examined using product inhibition, dead-end inhibition and alternate substrate experiments. The results are consistent with a steady-state random mechanism for the NAD-linked and an ordered, sequential mechanism with NADP+ binding first for the NADP-linked reaction. Thus, the enzyme can bind NADP+, NAD+, and glucose 6-phosphate, but the enzyme-glucose 6-phosphate complex can react only with NAD+, not with NADP+. This affects the rate equation for the NADP-linked reaction by introducing a term for a dead-end enzyme-glucose 6-phosphate complex. The kinetic mechanisms represent revisions of those proposed previously (C. Olive, M.E. Geroch, and H.R. Levy, 1971, J. Biol. Chem. 246, 2047-2057) and provide a kinetic basis for the regulation of coenzyme utilization of the enzyme by glucose 6-phosphate concentration (H.R. Levy, and G.H. Daouk, 1979, J. Biol. Chem. 254, 4843-4847) and NADPH/NADP+ concentration ratios (H.R. Levy, G.H. Daouk, and M.A. Katopes, 1979, Arch, Biochem. Biophys. 198, 406-413). The kinetic mechanisms were found to be the same at pH 6.2 and pH 7.8. The kinetics of ATP inhibition of the NAD- and NADP-linked reactions were examined at pH 6.2 and pH 7.8. The results are interpreted in terms of ATP addition to binary enzyme-coenzyme and enzyme-glucose 6-phosphate complexes.

Regulation of coenzyme utilization by the dual nucleotide-specific glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroids.

Abstract The dual wavelength assay technique (H. R. Levy, and G. H. Daouk, 1979, J. Biol. Chem.254, 4843–4847) is used to examine the rates of the NADP- and NAD-linked reactions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase simultaneously under various conditions. Inhibition by ATP, MgATP2−, acetyl-CoA, and palmitoyl-CoA is greatly diminished at high glucose 6-P concentration which favors the NAD-linked reaction. Increasing NADPH NADP + concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction. The selective effects of glucose 6-P and the NADPH NADP + concentration ratio, which cannot be detected by conventional assays, are explained in terms of the differing kinetic mechanisms for the NADP-linked and NAD-linked reactions previously described (C. Olive, M. E. Geroch, and H. R. Levy, 1971, J. Biol. Chem.246, 2047–2057) . It is proposed that these effects constitute the mechanism whereby the nucleotide specificity of the amphibolic glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides is regulated.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: ligand-induced conformational changes

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated by trypsin, chymotrypsin, pronase E, thermolysin, 4.0 M urea, and by heating to 49 degrees C. It is protected, to varying degrees, against all these forms of inactivation by glucose 6-phosphate, NAD+, and NADP+. When these ligands are present at 10 times their respective KD concentrations, protection by NAD+ or glucose 6-phosphate is substantially greater than protection by NADP+. A detailed analysis was undertaken of the protective effects of these ligands, at varying concentrations, on proteolysis of glucose-6-phosphate dehydrogenase by thermolysin. This study confirmed the above conclusion and permitted calculation of KD values for NAD+, NADP+, and glucose 6-phosphate that agree with such values determined by independent means. For NADP+, two KD values, 6.1 microM and 8.0 mM, can be derived, associated with protection against thermolysin by low and high NADP+ concentrations, respectively. The former value is in agreement with other determinations of KD and the latter value appears to represent binding of NADP+ to a second site which causes inhibition of catalysis. A Ki value of 10.5 mM for NADP+ was derived from inhibition studies. The principal conclusion from these studies is that NAD+ binding to L. mesenteroides glucose-6-phosphate dehydrogenase results in a larger global conformational change of the enzyme than does NADP+ binding. Presumably, a substantially larger proportion of the free energy of binding of NAD+, compared to NADP+, is used to alter the enzymes conformation, as reflected in a much higher KD value. This may play an important role in enabling this dual nucleotide-specific dehydrogenase to accommodate either NAD+ or NADP+ at the same binding site.

Glucose-6-phosphate dehydrogenase from rabbit erythrocytes

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) was purified from rabbit erythrocytes. Initial velocity studies and product and and dead-end inhibitor studies with this enzyme are consistent with a rapid equilibrium random mechanism with an enzyme-NADPH-glucose 6-phosphate dead-end complex.

Anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyl-transferase from Salmonella typhimurium: Inactivation of glutamine-dependent anthranilate synthetase by agarose-bound anthranilate

Exposure of the anthranilate synthetase-anthranilate phosphoribosyltransferase enzyme complex (chorismate pyruvate-lyase (amino-accepting) EC 4.1.3.27 and N-(5-phosphoribosyl)-anthranilate pyrophosphate phosphoribosyl-transferase, EC 2.4.2.18) from Salmonella typhimurium to agarose-bound anthranilate led to the slow inactivation of glutamine-dependent anthranilate synthetase activity, an activity dependent on protein-protein interaction in the enzyme complex. Region I of phosphoribosyltransferase, the location of the enzyme complex glutaminase activity, is the site of alteration. Phosphoribosyltransferase and NH3-dependent anthranilate synthetase activities and trypto phan regulation of phosphoribosyltransferase were unaffected by the anthranilate matrix. The molecular weight (280 000) and subunit molecular weight (62 000) of the enzyme complex eluted from an anthranilate matrix were identical to those of enzyme complex purified by classical methodology. The enzyme complex could be partially protected against inactivation by storiing in 0.1 M L-glutamine or 30% glycerol and completely protected by storing in 50% glycerol at -18 degrees C. Evidence is presented that the anthranilate matrix acts as a hydrophobic matrix and may be binding to and altering a hydrophobic region in the enzyme complex. The anthranilate matrix provides a convenient tool for altering a specific region of an enzyme complex without covalent modification. At the same time, the results demonstrate that affinity matrices are not necessarily innocuous but may subject macromolecules to an adverse environment not previously recognized.

Studies on the subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium☆

Abstract Both uncomplexed subunits of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium have an absolute requirement for divalent metal ions which can be satisfied by Mg 2+ , Mn 2+ , or Co 2+ . The metal ion kinetics for uncomplexed anthranilate synthetase give biphasic double-reciprocal plots and higher apparent K m values than those for anthranilate synthetase in the enzyme complex. In contrast, the apparent K m values for phosphoribosyltransferase are the same whether the enzyme is uncomplexed or complexed with anthranilate synthetase. This suggests that the metal ion sites on anthranilate synthetase, but not those on phosphoribosyltransferase, are altered upon formation of the enzyme complex. These results and the results of studies reported by others, suggest that complex formation between anthranilate synthetase and phosphoribosyltransferase leads to marked alterations at the active site of the former, but not the latter enzyme. Uncomplexed anthranilate synthetase can be stoichiometrically labeled with Co(III) under conditions which lead to inactivation of 75% of its activity. A comparison of the effects of anthranilate and tryptophan on phosphoribosyltransferase activity in the uncomplexed and complexed forms shows that anthranilate, but not tryptophan, inhibits the uncomplexed enzyme. The complexed phosphoribosyltransferase shows substrate inhibition by anthranilate binding to the phosphoribosyltransferase subunits. In contrast, in a tryptophan-hypersensitive variant complex, anthranilate inhibits phosphoribosyltransferase activity by acting on the anthranilate synthetase subunits. The data are interpreted to mean that there are two classes of binding sites for anthranilate, one on each type of subunit, which may participate in the regulation of anthranilate synthetase and phosphoribosyltransferase under different conditions.

The effects of 2H2O on mammary glucose-6-phosphate dehydrogenase

Abstract The NADP-linked and NAD-linked reactions catalyzed by rat mammary gland glucose-6-phosphate dehydrogenase are differentially affected by 2H2O. The enzyme is stabilized by 2H2O against inactivation in the cold at alkaline and neutral pH. These effects of 2H2O resemble those produced by glycerol and are attributed to the stabilization of Monomer X, the enzyme form possessing higher NAD-linked and lower NADP-linked activity than Monomer Y.