The role of the catalytic base in the protein tyrosine kinase Csk.
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 pKa. 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 kcat (approximately 10(4)) but relatively little effect on substrate Km values compared with wild-type Csk. Examination of the thio effect (kcat-ATP/kcat-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 gamma-phosphate of ATP toward electrophilic attack. These results may have significant impact on protein tyrosine kinase inhibitor design.
Cole
P A
PA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Grace
M R
MR
Phillips
R S
RS
Burn
P
P
Walsh
C T
CT
D000595:Amino Acid Sequence; D000081247:CSK Tyrosine-Protein Kinase; D002384:Catalysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry; D014783:Viscosity; D019061:src-Family Kinases
D010455:Peptides; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases