KINETIC-ANALYSIS OF A PROTEIN-TYROSINE KINASE REACTION TRANSITION-STATE IN THE FORWARD AND REVERSE DIRECTIONS

Protein tyrosine kinases catalyze the transfer of the gamma-phosphoryl ,group from ATP to tyrosine residues in proteins and are important enz ymes in cell signal transduction. We have investigated the catalytic p hosphoryl transfer transition state of a protein tyrosine kinase react ion catalyzed by Csk by analyzing a series of fluorotyrosine-containin g peptide substrates. It was established for five such fluorotyrosine- containing peptide substrates that there is good agreement between the tyrosine analogue phenol pK(a) and the ionizable group responsible fo r the basic limb of a pH rate profile analysis. This indicates that th e substrate tyrosine phenol must be neutral to be enzymatically active . Taken together with previous data indicating a small beta(nucleophil e) coefficient (0-0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In addition, the beta(leavin g group) coefficient was measured for the reverse protein tyrosine kin ase reaction and shown to be -0.3. This value is in good agreement wit h a previously reported nonenzymatic model phosphoryl transfer reactio n carried out under acidic conditions (pH 4) and is most readily expla ined by a transition state with significant proton transfer to the dep arting phenol.