Inhibition of glucose 6-phosphate dehydrogenase by adenosine 5'-triphosphate.

D-glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the oxidation of glucose 6-phosphate (G6P) to 6-phosphogluconate-6-lactone and thereby constitutes the step which introduces hexoses into the "pentose cycle" pathway. This well-known enzyme, which is widely distributed in nature, was purified to varying degrees from different sources (for literature see refs. 1 and 2). The yeast enzyme,3 4 which is now commercially available in a high degree of purity, is extensively used for analytical purposes in biochemical research, especially for the assay of ATP, NADP, hexoses, sugar phosphates, and several kinases. While employing yeast G6PDH in a coupled reaction for the analysis of a bacterial hexokinase-ATP system, we have noticed some inhibition of the expected initial rate of NADPH formation when compared to that observed in control systems with G6P. A search for the cause of this phenomenon has indicated that ATP exerted an inhibitory effect on the reaction catalyzed by G6PDH. This observation is described in the present communication. Materials and Methods.-Crystalline yeast G6PDH and hexokinase were obtained from Boehringer und Soehne, GmbH. G6P, NADP, and ATP were preparations supplied by Boehringer and by Sigma Chemical Co. Nucleotides, sugar phosphates, and other biochemical reagents were purchased from Calbiochem, Boehringer, and Pabst Laboratories Biochemicals, Inc. NADPH formation in the G6PDH reaction was followed at 340 my using quartz cells with a 10-mm light path in the Gilford model 2000 multiple-sample automatic recording spectrophotometer, temperature controlled at 250 or 300. Reaction was usually initiated by the addition of NADP as the last component. In G6PDHhexokinase-coupled systems, the reaction was started by the addition of hexokinase. A unit of enzyme activity was defined as that amount of enzyme which produced one Amole of NADPH/min at Vmax conditions at the pH in which the particular set of experiments was performed. Results.-Inhibition by A TP: Inclusion of ATP in a standard G6PDH reaction system resulted in a significant inhibitory effect on the rate of oxidation which was also pH-dependent (Fig. 1). It is clear that the inhibition is much more pronounced at neutral pH values, whereas it is not so apparent between pH 8.0 and 8.5, the range usually employed for the assay of this enzyme system. Inhibition by ATP could be relieved competitively with G6P (Fig. 2). M\g++ strongly diminished the inhibition by ATP (Fig. 3). It was found that maximum change in the magnitude of inhibition by ATP occurred with about an equimolar concentration of Mg++ (Fig. 3). Similar to what has been observed by Glaser and Brown,3 Mg++ did not change appreciably the Vmina of the reaction at pH 7.3, but caused a slight change in the apparent Km for G6P from 4.0 X 10-5 M to 7.0