Evaluation of the catalytic mechanism of recombinant human Csk (C-terminal Src kinase) using nucleotide analogs and viscosity effects.
Tyrosine kinases catalyze phosphoryl transfers from ATP to tyrosine residues in proteins. Despite their growing importance, their kinetic mechanism has remained largely unexplored. In this study, we have investigated the tyrosine kinase reaction catalyzed by purified human recombinant Csk (C-terminal Src kinase). Poly(Glu,Tyr) 4:1 was used as the tyrosine-containing substrate. Both ATP and poly(Glu,Tyr) were shown to be well behaved saturable substrates for recombinant Csk, with Km values that were in reasonable agreement with literature values reported for the non-recombinant enzyme and with kcat about 40 min-1. A sequential kinetic mechanism is suggested by a steady state kinetic analysis. Inhibitor studies with ADP and beta,gamma-imidoadenosine 5'-triphosphate were performed, and these results provided evidence against the possibility that ordered binding of peptide prior to ATP occurs. While a suitable competitive inhibitor of poly(Glu,Tyr) has not yet been identified, other evidence pointed to a rapid equilibrium random mechanism. Csk utilized adenosine 5'-O-(3-thiotriphosphate) in place of ATP. The phosphorothioyl transfer occurred with a kcat about 15-20-fold lower than the ATP reaction but with similar Km values. Deuterium solvent isotope effects on kcat were small for both reactions in a pH-independent range, consistent with the possibility that proton transfer is asymmetric in the reaction transition state. Using viscosity effects, ADP product release was suggested to be partially rate determining for catalysis in the standard ATP reaction. A comparison of the Csk kinetic mechanism with that of protein kinase A is discussed.
Cole
P A
PA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Burn
P
P
Takacs
B
B
Walsh
C T
CT
D000595:Amino Acid Sequence; D000081247:CSK Tyrosine-Protein Kinase; D002384:Catalysis; D006801:Humans; D007554:Isotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014783:Viscosity; D019061:src-Family Kinases
D007554:Isotopes; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C000631346:CSK protein, human