The role of the catalytic base in the protein tyrosine kinase Csk

Abstract

A potential distinguishing feature between protein tyrosine kinases and homologous serine/threonine kinases is the function of the catalytic base in these enzymes. In this study, we show that a peptide containing the unnatural amino acid trifluorotyrosine shows remarkably similar efficiency as a substrate of the tyrosine kinase Csk (C-terminal Src kinase) compared with the corresponding tyrosine-containing peptide despite a 4-unit change in the phenolic pK(a). These results argue against the importance of early tyrosine deprotonation by a catalytic base in Csk. To further explore the role of the proposed catalytic base, the Csk mutant protein D314E was produced. This mutant displayed a significant reduction in k(cat) (approximately 104) but relatively little effect on substrate K(m) values compared with wild-type Csk. Examination of the thio effect (k(cat)-ATP/k(cat)-adenosine 5'-O- (thiotriphosphate)) for D314E Csk led to the suggestion that a role of aspartate 314 may be to enhance the reactivity of the γ-phosphate of ATP toward electrophilic attack. These results may have significant impact on protein tyrosine kinase inhibitor design.