Kinetic analysis of 6-phosphogluconate dehydrogenase from bass liver: effects of temperature and pH on its catalytic...

Kinetic studies of the reaction between 6-phosphogluconate and NADP catalyzed by purified 6-phosphogluconate dehydrogenase from bass liver were made at pH 7.5 in Tris-HCl buffer. Relationships among the initial rate coefficients for 6-phosphogluconate, NADP, and Mg2+ suggest that the addition of Mg2+ and NADP to 6-phosphogluconate dehydrogenase does not follow an obligatory order, probably being random, in which 6-phosphogluconate combines second with the enzyme. The Michaelis constants for NADP, 6-phosphogluconate, and Mg2+ are 0.88 microM, 26.66 microM, and 3.33 mM, respectively. pK values for the enzyme-6-phosphogluconate and enzyme-NADP complexes have been obtained. The variations in the true Km values for NADP with pH are only threefold at most, and seem to be not especially significant. A plausible explanation for the physiological significance of the influence of temperature on the Km values is given. Kinetic studies show that phosphoenolpyruvate is a noncompetitive inhibitor of the enzyme with respect to 6-phosphogluconate and a competitive inhibitor with respect to NADP with Ki values of 0.54 mM against 6-phosphogluconate and 0.15 mM against NADP.