AZOTOBACTER-VINELANDII GLUCOSE-6-PHOSPHATE-DEHYDROGENASE PROPERTIES OF NAD-LINKED AND NADP-LINKED REACTIONS

Glucose 6-phosphate oxidation, catalyzed by purified Azotobacter vinel andii glucose 6-phosphate dehydrogenase, was studied with respect to t he selective utilization of NAD, NADP, thionicotinamide adenine dinucl eotide or thionicotinamide adenine dinucleotide phosphate as coenzyme. A sigmoidal relationship was observed for the effect of substrate con centration on initial velocities when either NAD, NADP or thionicotina mide adenine dinucleotide was used as coenzyme, with N values from the Hill equation equalling 2.0, 1.7, and 1.7, respectively. The thionico tinamide analogs of NAD and NADP both functioned as coenzyme-competiti ve inhibitors of the enzyme-catalyzed NAD- and NADP-linked reactions. A dual wavelength assay, using a combination of NADP and thio-NAD, was established and wets used to demonstrate that increasing glucose 6-ph osphate concentration did not change the enzyme preference for the coe nzyme form used. Sigmoidal relationships were observed for reduction o f both dinucleotides, and N values were the same as those observed whe n each dinucleotide was studied as the only coenzyme form present in r eaction mixtures. Using the dual wavelength assay, inhibition by isoci trate, 6-phosphogluconate, ATP, and palmitoyl-CoA was shown to be equa lly effective in both NAD- and NADP-linked reactions. An enzyme activa tor, glucosamine 6-phosphate, altered the glucose 6-phosphate sigmoidi city through activation at low substrate concentrations. (C) 1997 Else vier Science B.V.