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 re sidues in proteins. Despite their growing importance, their kinetic me chanism has remained largely unexplored. In this study we have investi gated the tyrosine kinase reaction catalyzed by purified human recombi nant Csk (C-terminal Src kinase). Poly(Glu,Tyr) 4:1 was used as the ty rosine containing substrate. Both ATP and poly(Glu,Tyr) were shown to be well behaved saturable substrates for recombinant Csk, with K-m val ues that were in reasonable agreement with Literature values reported for the non-recombinant enzyme and with k(cat) about 40 min(-1). A seq uential kinetic mechanism is suggested by a steady state kinetic analy sis. Inhibitor studies with ADP and beta,gamma-imidoadenosine 5'-triph osphate were performed, and these results provided evidence against th e possibility that ordered binding of peptide prior to ATP occurs. Whi le a suitable competitive inhibitor of poly(Glu,Tyr) has not yet been identified, other evidence pointed to a rapid equilibrium random mecha nism. Csk utilized adenosine 5'-O-(3-thiotriphosphate) in place of ATP . The phosphorothioyl transfer occurred with a k(cat) about 15-20-fold lower than the ATP reaction but with similar K-m values. Deuterium so lvent isotope effects on k(cat) were small for both reactions in a pH- independent range, consistent with the possibility that proton transfe r is asymmetric in the reaction transition state. Using viscosity effe cts, ADP product release was suggested to be partially rate determinin g for catalysis in the standard ATP reaction. A comparison of the Csk kinetic mechanism with that of protein kinase A is discussed.