Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalys is. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and p urified as fusion proteins of glutathione-S-transferase from Escherichia co li and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073 F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to t he wild-type enzyme, respectively. For all mutant enzymes, the K-m values f or ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compar ed to the wild-type enzyme. The slopes for the plots of relative turnover v ersus solvent viscosity [(k(cat))(eta)] are 0.71 +/- 0.08, 0.10 +/- 0.06, a nd approximate to 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The di ssociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a com petitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q c ompared to wild type. Y1073E and Y1073Q are strongly activated by free magn esium to the same extent and the apparent affinity constant for the metal i s similar to that for the wild-type enzyme. The data indicate that the majo r role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-s ite accessibility or the local environment of the activating metal. Finally , the similar rate enhancements for phosphoryl transfer in v-Fps compared t o protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon au tophosphorylation suggest a conserved mechanism for communication between t he activation loop and the catalytic residues of these two enzymes.