Proton demand inversion in a mutant protein tyrosine kinase reaction.
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 beta(nuc), 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.
Williams
Daniel M
DM
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Room 316, Hunterian Building, 725 North Wolfe Street, Baltimore, Maryland 21205, USA.
Cole
Philip A
PA
D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D011522:Protons; D013379:Substrate Specificity
D009842:Oligopeptides; D011522:Protons; D001224:Aspartic Acid; C086603:Matk protein, mouse; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)