These results provide useful practical information for chemists and biochemists who may wish to employ this new cross-linking chemistry for the analysis of protein complexes. They also shed new light on the mechanism of this interesting reaction.

Protein tyrosine kinases catalyze the transfer of the γ-phosphoryl group from ATP to tyrosine residues in proteins and are important enzymes in cell signal transduction. We have investigated the catalytic phosphoryl transfer transition state of a protein tyrosine kinase reaction catalyzed by Csk by analyzing a series of fluorotyrosine-containing 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 for the basic limb of a pH rate profile analysis. This indicates that the substrate tyrosine phenol must be neutral to be enzymatically active. Taken together with previous data indicating a small βnucleophile coefficient (0−0.1), these results strongly support a dissociative transition state for phosphoryl transfer. In addition, the βleaving  group coefficient was measured for the reverse protein tyrosine kinase reaction and shown to be −0.3. This value is in good agreement with a previously reported nonenzymatic model phosphoryl transfer reaction carried out under acidic conditions (pH 4) and is most readily explained by a transition state with significant proton transfer to the departing phenol.

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