Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps

Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v -Fps enhances the phosphoryl transfer reaction without influencing substrat e, ATP, or metal ion binding affinities [Saylor, P., et al. (1998) Biochemi stry 37, 17875-17881]. A structural model of v-Fps, generated from the insu lin receptor, indicates that pTyr-1073 chelates two arginines. Mutation of these residues to alanine (R1042A and R1066A) results in weakly phosphoryla ted enzymes, indicating that one electropositive center is insufficient for attaining maximum loop phosphorylation and concomitant high catalytic acti vity. While the turnover rate for R1066A is similar to that for a mutant la cking a phosphorylatable residue in the activation loop, the rate for R1042 A is 50-fold slower. While solvent perturbation studies suggest that the fo rmer is due to a slow phosphoryl transfer step, the latter effect results f rom a slow conformational change in the mutant, potentially linked to motio ns in the catalytic loop. Binding of a stoichiometric quantity of Mg2+ is e ssential for ATP binding and catalysis, while binding of an additional Mg2 ion activates further the wild-type enzyme. The affinity of the R1066A enz yme for the second Mg2+ ion is 23-fold higher than that of the phosphorylat ed or unphosphorylated form of wild-type v-Fps, with substrate binding unaf fected. Conversely, the affinity of R1066A for a substrate mimic lacking a phosphorylation site is 12-fold higher than that for the phosphorylated or unphosphorylated form of wild-type v-Fps, with binding of the second Mg2+ i on unaffected. A comparison of these enzyme-independent parameters indicate s that Arg-1042 and Arg-1066 induce strain in the active site in the repres sed form of the enzyme. While this strain is not relieved in the phosphoryl ated form, the improvements in catalysis in activated v-Fps compensate for reduced metal and substrate binding affinities.