Crystalline D -Gluconate 6-Phosphate Dehydrogenase"

A method for t.he purification of n-gluconate 6-phosphate dchydrogenase from brewers' yeast has been described by Horecker and Smyrniotis. (1). The enzyme preparation also catalyzed the oxidation of n-glucose 6-phosphate and isocitrate and in addition contained significant amounts of n-ribose 5-phosphate isomerasc. The utility of the enzyme for the assay of n-gluconate 6-phosphato and for the preparation of n-ribulose 5-phosphate was seriously affected by the prcsencc of thcsc contaminating activitics. For a further investigation of the properties of the enzyme and of the nature of the two-step reaction, a more highly purified preparation was necessary. A method has now been developed which yields from Candida yeast a crystalline preparation which is free of detectable quantitics of I)-glucose 6-phosphata dchydrogenase and r)-ribose 5-phosphate isomerasc. To remove the former enzyme, advantage was taken of its lability to heat after treatment with charcoal (2, 3). n-Glucose 6-phosphate dchydrogcnasc is known to bc unstable in t,he absence of triphosphopyridine nuclcotidc. With the purified enzyme, only n-ribulosc 5-phosphate is formed, and this ester can thus be obtained free of n-ribose 5-phosphate and n-xylulose j-phosphate. Since the crystalline enzyme preparation catalyzes the osidation of reduced triphosphopyridine nucleotide by CO, and o-ribulose phosphate at a rate equal to that observed with crude enzyme preparations, wc have concluded that a single enzyme is rcsponsible for both oxidation and decarboxylation reactions. We have also undertaken an investigation of the specificity of the Mg++ requirement (1) and havn found that at high concentrations of NaCl, full activity is obtained in the absence of Mg++. There is reason to believe that the nature of t,he anion plays a role in the activation of the enzyme