Kinetics of the Reaction of Baker′s Yeast Glucose-6-Phosphate-Dehydrogenase with 5,5′-Dithiobis(2-Nitrobenzoic Acid)

Abstract

The kinetics of the reaction of baker′s yeast glucose-6-phosphate dehydrogenase with excess 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were studied at pH 8.5 and 30°C and at constant ionic strength of 0.01 and in the absence and in the presence of NADP + or glucose 6-phosphate. The reaction follows pseudo-first-order kinetics irrespective of whether these substrates are absent or present. The observed pseudo-first-order rate constant is reduced in the presence of NADP + or glucose 6-phosphate but on a molar basis, glucose 6-phosphate is more effective than NADP + in protecting the sulfhydryl groups of the enzyme against the reaction with DTNB. In the presence of NADP +, the observed pseudo-first-order rate constant decreases to a constant, but finite, value at a saturating coenzyme concentration. The effect of NADP + on the rate constant is consistent with the presence of noninteracting coenzyme binding sites in baker′s yeast glucose-6-phosphate dehydrogenase.