Kinetic mechanism of glucose-6-phosphate dehydrogenase from the lactating rat mammary gland. Implications for regulation.

The steady state kinetic mechanisms for glucose-6-P dehydrogenase from lactating rat mammary glands were derived for the NADP-linked and NAD-linked reactions. Initial velocity studies and inhibition patterns using NADPH and glucosamine-6-P are consistent with sequential mechanisms with random order addition of glucose-6-P and coenzyme. No evidence was found for enzyme isomerization or dead-end complexes. Both NADP+ and glucose-6-P were found to quench the fluorescence of the enzyme. The dissociation constant for NADP+ derived from fluorescence quenching titrations was similar to the kinetically derived binding constant, consistent with random order binding. Analysis of fluorescence quenching by glucose-6-P revealed the presence of two classes of binding sites on the enzyme with different affinities for the substrate. Isotope effects were determined using both deuterium and tritium. From these it was concluded that the transfer of hydrogen from glucose-6-P to NADP+ limits the overall reaction rate by only 15 to 19%. We conclude that the mechanism for the reaction, with either NADP' or NAD+ as coenzyme, is probably partial rapid equilibrium random. Such a mechanism would be advantageous for mammary glucose-6-P dehydrogenase because it would permit glucose-6-P to counteract NADPH inhibition. Support for this idea was provided by experiments in which increased concentrations of glucose-6-P partially reversed the inhibition f purified mammary glucose-6-P dehydrogenase at high NADPH/ NADP+ ratios.