ISOCITRATE DEHYDROGENASE KINASE PHOSPHATASE - KINETIC CHARACTERISTICSOF THE WILD-TYPE AND 2 MUTANT PROTEINS/

Isocitrate dehydrogenase (IDH) of Escherichia coti is regulated by a b ifunctional protein, IDH kinase/phosphatase. In addition to the kinase and phosphatase activities, this protein catalyzes an intrinsic ATPas e reaction. The initial velocity kinetics of these activities exhibite d extensive similarities, IDH kinase and phosphatase both yielded inte rsecting double-reciprocal plots. In addition, we observed similar val ues for the kinetic constants describing interactions of the kinase an d phosphatase with their protein substrates and the interactions of al l three activities with ATP, In contrast, while the maximum velocities of IDH kinase and IDH phosphatase were nearly equal, they were 10-fol d less than the maximum velocity of the ATPase. Although the IDH phosp hatase reaction required either ATP or ADP, it was not supported by th e nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate. The kinet ic properties of wild-type IDH kinase/phosphatase were compared with t hose of two mutant derivatives of this protein, The mutations in these proteins selectively inhibit IDH phosphatase activity. Inhibition of IDH phosphatase resulted from three factors: decreases in the maximum velocities, reduced affinities for phospho-IDH, and a loss of coupling between ATP and phospho-IDH, These mutations also affected the proper ties of IDH kinase, increasing the maximum velocities and decreasing t he affinities for ATP and phospho-IDH. The intrinsic ATPase activities also exhibited reduced affinity for ATP, These results are discussed in the context of a model which proposes that all three activities occ ur at the same active site.