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Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD+ or NADP+ as coenzyme. Kinetic studies showed that NAD+ and NADP+ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057) . In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP+ and 50% for NAD+. The dissociation constant for NADP+, determined from fluorescence quenching measurements, is 3 μ m , which is similar to the previously determined Km of 5.7 μ m and Ki of 5 μ m . The dissociation constant for NAD+ is 2.5 m m , which is 24 times larger than the previously determined Km of 0.106 m m . Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD+ but not for NADP+. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD+ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD+; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μ m ; the previously determined Ki is 37.6 μ m . The dissociation constant for NAD′ is 2.8 m m , in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP+-competitive and NAD+-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD+ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure. |
