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R E Viola

Publications and source records attributed to R E Viola.

47 records · Page 3Linked to original sources
PubMed1984

Kinetic studies of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: pH variation of kinetic parameters.

The specificity and kinetic parameters of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been examined under a range of conditions in order to elucidate details about the mechanism of action of this enzyme. The rate of oxidation of glucose 6-phosphate is inhibited by the addition of various organic solvents. However, the low, inherent glucose dehydrogenase activity of this enzyme was stimulated under these conditions, and was further activated by divalent anions that were observed to be inhibitors of the glucose 6-phosphate dehydrogenation. From an examination of the pH variation of the enzyme kinetic parameters two groups on the enzyme that appear to be involved in the binding of the phosphate group of the sugar substrate have been detected. An enzyme catalytic group, probably a carboxylic acid, has been identified that accepts the proton from the hydroxyl group at carbon-1 of the sugar substrate during its oxidation to a lactone. The ionization of a group on the enzyme with a pK of 8.7 resulted in an increase in the maximum velocity of the glucose-6-phosphate dehydrogenase activity of the enzyme as a consequence of a pH-dependent product release step that is no longer rate limiting at high pH. Stabilization of gluconic acid-delta-lactone against nonenzymatic hydrolysis by organic solvents has allowed the kinetic parameters of the reverse reaction to be reliably measured for the first time in a narrow pH range.

Anions↗
PubMed1984

A multinuclear NMR relaxation study of the interaction of divalent metal ions with L-aspartic acid.

Carbon-13 spin-lattice relaxation times, T1, have been measured for aqueous solutions of L-aspartic acid, L-alanine, O-phospho-L-serine, and 2-mercapto-L-succinic acid in the presence of the paramagnetic metal ions, Cu2+ and Mn2+, and Mg2+ as a diamagnetic control, at ambient temperature and neutral pH. Nitrogen-15, oxygen-17 and proton relaxation times were also obtained for L-aspartic acid and phosphorus-31 relaxation times for O-phospho-L-serine under similar conditions. The structures of these complexes in solution were determined from the various metal ion-nuclei distances calculated from the paramagnetically-induced relaxation. These results indicate that the Cu2+ interaction with L-aspartic acid is through alpha-amino and beta-carboxyl groups while Mn2+ coordinates most strongly through alpha- and beta-carboxyl groups, with the possibility of a weak interaction through the amino group. An examination of the coordination of these divalent metal ions to an analog of L-aspartic acid in which the beta-carboxyl group is replaced by a phosphate group (O-phospho-L-serine) indicated that Cu2+ coordination is now probably through the alpha-amino and phosphate groups, while this analog is a monodentate ligand for Mn2+ coordinating through the phosphate group. Removal of the beta-carboxyl group (L-alanine) also results in Cu2+ coordination through the alpha-carboxyl and alpha-amino groups, and the same ligand interactions are observed with Mn2+. Replacement of the alpha-amino group of L-aspartic acid with an -SH group (2-mercapto-L-succinate) is sufficient to eliminate any specific coordination with either Cu2+ or Mn2+.

Alanine↗
PubMed1983

Solvent proton relaxation studies of cytochrome c oxidase solutions.

The interaction of solvent water protons with the bound paramagnetic metal ions of beef heart cytochrome c oxidase has been examined. The observed proton relaxation rates of enzyme solutions had a negative temperature dependence, indicating a rapid exchange between solvent protons in the coordination sphere of the metal ions and bulk solvent. An analysis of the dependence of the proton relaxation rate on the observation frequency indicated that the correlation time, which modulates the interaction between solvent protons and the unpaired electrons on the metal ions, is due to the electron spin relaxation time of the heme irons of cytochrome c oxidase. This means that at least one of the hemes is exposed to solvent. The proton relaxation rate of the oxidized enzyme was found to be sensitive to changes in ionic strength and to changes in the spin states of the metal ions. Heme a3 was found to be relatively inaccessible to bulk solvent. Partial reduction of the enzyme caused a slight increase in the relaxation rate, which may be due to a change in the antiferromagnetic coupling between two of the bound paramagnetic centers. Further reduction resulted in a decreased relaxation rate, and the fully reduced enzyme was no longer sensitive to changes in ionic strength. The binding of cytochrome c to cytochrome c oxidase had little effect on the proton relaxation rates of oxidized cytochrome oxidase indicating that cytochrome c binding has little effect on solvent accessibility to the metal ion sites.

Animals↗
PubMed1982

Substrate synergism and the kinetic mechanism of yeast hexokinase.

Michaelis constants for MgATP with yeast hexokinase vary from 28 microM with D-mannose to above 4 mM for the slow ATPase reaction, with the different values reflecting the degree of synergism in binding of MgATP and the sugar substrate. The best substrates show the greatest synergism, but the correlation is not exact. Similar synergistic binding between MgADP or its methylene analogue and phosphorylated sugars is seen. Product inhibiton of MgADP vs. MgATP and vice versa appears noncompetitive at low levels of variable substrate but becomes competitive at high levels. These patterns show that MgATP can combine with E-glucose-6-P (Ki = 4 mM) and MgADP with E-glucose (Ki = 1.6 mM). Isotope partitioning studies with glucose or glucose-6-P have determined the rates of release of these substrates from binary and ternary complexes and, together with reverse isotope exchange studies and the product inhibition studies mentioned above, have shown that the kinetic mechanism is a somewhat random one in which dissociation of sugars from productive ternary complexes is very slow, but release from nonproductive ternary complexes occurs at rates similar to those from binary enzyme-sugar complexes. D-Arabinose-5-P has a Km of 4.6 mM and a Vmax 5% that for glucose-6-P, confirming that the high Km for D-arabinose in the forward direction is caused by the low proportion in the furanose form. The dissociation constant of MgADP in the absence of sugars was determined from the Ki of 5.8 mM for MgADP as a competitive inhibitor vs. MgATP of the slow ATPase reaction.

Adenosine Diphosphate↗
PubMed1980

Interaction of metal(III)-adenosine 5'-triphosphate complexes with yeast hexokinase.

In the presence of glucose, yeast hexokinase is specifically and strongly inhibited by all MIIIATP (M = metal) complexes that do not hydrolyze at neutral pH, as long as the ionic radius of the metal is less than 0.89 A. Ki values vary from the micromolar range (0.16 microM for AlATP at pH 7, for example) to as low as 13 nM for LuATP. With glucose and fructose, the tightly bound complexes also show reversible, slow binding behavior, but with poor substrates, little or no change in inhibition constant with time is observed. The kinetics of citrate as an activator of the hexokinase reaction are consistent with its reaction with AlATP present as a contaminant in commercial ATP to form Al citrate. The complex of Al(III) with citrate is 5 orders of magnitude more stable than AlATP, whose Kd is 0.7 microM at pH 7. ATP that has been treated with excess EDTA and adsorbed on and eluted from charcoal is free of aluminum, and citrate no longer affects the kinetics of the hexokinase reaction. Glycerokinase is also specifically inhibited by trivalent metal ATP complexes (Ki = 4 microM at pH 7 for AlATP).

Adenosine Triphosphate↗
PubMed1980

Aldehyde-induced adenosine triphosphatase activities of fructose 6-phosphate and fructose kinases.

Chitose-6-P (2,5-anhydromannose-6-P) induces ATPase activity of fructose-6-P kinase with a Vmax 2-3% that of the normal kinase reaction with fructose-6-P or 2,5-anhydromannitol. Chitose (and presumably also chitose-6-P) is 52% hydrated in water while chitose deuterated at C-1 is 60% hydrated because of the equilibrium isotope effect of 0.73 on aldehyde hydration. Deuterated chitose-6-P gave a normal isotope effect on V/K of 1.23, but no effect on Vmax, showing that the free aldehyde is the activator and the hydrated form does not bind appreciably. With fructokinase, chitose can act either as a substrate, being phosphorylated at C-6 when adsorbed with C-6 next to MgATP, or as an inducer of ATPase activity when adsorbed with C-1 next to MgATP. The ATPase has a rate about 25% that of the kinase.

Adenosine Triphosphatases↗