Proton demand inversion in a mutant protein tyrosine kinase reaction

Abstract

In contrast to previous studies that have shown that the neutral phenol serves as the nucleophile for WT Csk-promoted phosphorylation of a tyrosine-containing substrate, the phenolate ion acts as primary nucleophile for the D314N Csk-catalyzed reaction. Rate comparisons of D314N Csk-promoted phosphotransfer using a series of fluorotyrosine-containing peptide substrates reveal a near zero βnuc, consistent with a dissociative mechanism of phosphotransfer. These combined results argue against a hydroxy nucleophile-to-phosphate proton transfer occurring prior to an associative transition state of phosphoryl transfer. Copyright © 2002 American Chemical Society.