RATE-DETERMINING STEPS FOR TYROSINE PHOSPHORYLATION BY THE KINASE DOMAIN OF V-FPS

The rate-determining steps in the phosphorylation of four tyrosine-con taining peptides by the kinase domain of the nonreceptor tyrosine prot ein kinase v-fps were measured using viscosometric methods. The peptid es were phosphorylated by a fusion protein of glutathione-S-transferas e and the kinase domain of v-fps (GST-kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), Il (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with s imilar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on k(cat) and no effect on k(cat)/K -peptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple th ree-step mechanism involving substrate binding, phosphoryl group trans fer, and net product release. Two competitive inhibitors (EAEIFEAIE an d DADIFDAID) exhibited Kr values that are 6-10-fold higher than the K- peptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that k(cat) is partially limited by both phosphoryl group transfer (40-100 s(-1)) and product release (17-22 s(-1)). GST-kin phosphorylates pepti de IV (R(5)AENLEYamide) with a low K-m (100 mu M) and a k(cat) that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either k(cat) or k(cat)/K-peptide. This suggests that highly viscous solutions do n ot perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kin ase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-s pecific cAMP-dependent protein kinase and its best substrate LRRASLG ( Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar a ffinity for their substrates and both enzymes release their products a t a similar rate. This implies that the differences in catalytic effic iency between serine- and tyrosine-specific protein kinases lie exclus ively in the rate constants for phosphoryl group transfer and not in s ubstrate absorption or product desorption.