Regulation of coenzyme utilization by bovine liver glutamate dehydrogenase: investigations using thionicotinamide analogues of NAD and NADP in a dual wavelength assay.
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.
D000818:Animals; D002417:Cattle; D003067:Coenzymes / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives* / Q000494:pharmacology; D009249:NADP / Q000031:analogs & derivatives* / Q000494:pharmacology; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D003067:Coenzymes; D009243:NAD; C015541:thionicotinamide adenine dinucleotide phosphate; C003451:thionicotinamide adenine dinucleotide; D009249:NADP; D005969:Glutamate Dehydrogenase