thionicotinamide-adenine-dinucleotide and thionicotinamide-adenine-dinucleotide-phosphate

Azotobacter vinelandii glucose-6-phosphate dehydrogenase isolated from cell sonicates was purified 81-fold to electrophoretic homogeneity and a specific activity of 73 units/mg protein using ion-exchange and Matrex Dye chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular exclusion chromatography indicated the enzyme to be a tetramer composed of 52,000 M(r) subunits. The enzyme utilized both NAD and NADP as coenzymes with Km values of 220 and 50 microM, respectively. In addition, the purified enzyme functioned well with the thionicotinamide analogs of NAD and NADP. A sigmoidal response was observed in studies of the effect of glucose 6-phosphate concentration on initial velocities. Evidence in support of one enzyme with dual coenzyme specificity was obtained in purification, thermodenaturation, and inhibitor studies. The enzyme exhibited a pH optimum of 8.5. Coenzyme-competitive inhibition was observed with nine adenosine derivatives with no significant selectivity shown for 2'-phosphoryl derivatives. Ki values for product inhibition by NADH and NADPH were higher than the Km values for the respective oxidized forms of the coenzymes.

Topics: Adenine Nucleotides; Azotobacter vinelandii; Binding, Competitive; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Glucosephosphate Dehydrogenase; Kinetics; Macromolecular Substances; Molecular Weight; NAD; NADP; Substrate Specificity

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.

Topics: Adenosine Triphosphate; Animals; Fishes; Kinetics; Malate Dehydrogenase; Mitochondria, Muscle; NAD; NADP; Spectrophotometry

1. The coenzyme preference of bovine liver glutamate dehydrogenase (GDH) was probed using dual wavelength spectroscopy and pairing the thionicotinamide analogues, S-NAD or S-NADP (which have absorbance maxima at 400 nm), with the natural coenzymes, NADP or NAD. 2. S-NAD and S-NADP were found to be good alternate substrates for GDH: the apparent Km's for the thioderivatives were similar to those of the corresponding natural coenzymes, the apparent Km's for glutamate were unaltered by the substitution of the thioderivatives, and the effects of inhibitors and activators on S-NAD or S-NADP kinetics were qualitatively the same as those found for NAD or NADP, respectively. 3. Dual wavelength assays paired NAD and S-NADP or S-NAD and NADP to study the simultaneous reduction of the two coenzymes. Conditions of increasing glutamate concentrations produced differential effects on the rates of the NAD vs NADP reactions, the result, with either nucleotide pair, promoting the NADP linked reaction. 4. Activators and inhibitors of the GDH reaction also showed differential effects upon the NAD vs NADP linked reaction rates in the dual wavelength assay. ADP and leucine, which activate both the NAD and the NADP linked reactions in single coenzyme assays, preferentially activate the NADP or S-NADP linked reactions in the dual nucleotide assays. GTP produced greater inhibition of the NAD or S-NAD linked reactions than of the NADP or S-NADP reactions while ATP inhibited NAD or S-NAD reactions and activated NADP or S-NADP reactions. The net effect of all metabolite modulators was to promote the NADP linked reaction by decreasing the activity ratios, v(NAD)/v(S-NADP) or v(S-NAD)/v(NADP). 5. The results are consistent with the suggestion that NADP is the preferred coenzyme for the oxidative deamination of glutamate by GDH even though the enzyme is capable of utilizing either coenzyme in vitro.

Topics: Animals; Cattle; Coenzymes; Enzyme Activation; Glutamate Dehydrogenase; Liver; NAD; NADP; Spectrophotometry; Substrate Specificity

The thionicotinamide analogues of NAD+ and NADP+ were shown to be good alternative coenzymes for bovine glutamate dehydrogenase, with similar affinity and approx. 40% of the maximum velocity obtained with the natural coenzymes. Both thionicotinamide analogues show non-linear Lineweaver-Burk plots, which with the natural coenzymes have been attributed to negative co-operativity. Since the reduced thionicotinamide analogues have an isosbestic point at 340nm and have an absorption maximum at 400nm, it is possible to monitor reduction of natural coenzyme and thionicotinamide analogue simultaneously by dual-wavelength spectroscopy. When glutamate dehydrogenase is presented with NADP+ and thio-NADP+ simultaneously, the enzyme oligomer senses saturation of its coenzyme-binding sites irrespective of the exact nature of the coenzyme and locks the oligomer into its highly saturated form even when low saturation of the monitored coenzyme is present. These experiments substantiate the suggestion that glutamate dehydrogenase shows negative co-operativity in its catalytically active form.

Topics: Animals; Binding Sites; Cattle; Deamination; Glutamate Dehydrogenase; Glutamates; Kinetics; NAD; NADP; Spectrophotometry; Thionucleotides