DIVALENT ION EFFECTS AND INSIGHTS INTO THE CATALYTIC MECHANISM OF PROTEIN-TYROSINE KINASE CSK

Csk (C-terminal Src kinase) is a protein tyrosine kinase which catalyz es the transfer of the gamma-phosphoryl group of ATP to the tyrosine h ydroxyl of proteins in the presence of a divalent ion. Previous work w ith poly(Glu,Tyr) as the tyrosine-containing substrate and Mn as the d ivalent ion defined a ternary complex mechanism with ADP product relea se partially rate-determining [Cole, P. A., et al. (1994) J. Biol. Che m. 269, 30880-30887]. In this current study, ionic strength and divale nt ion effects were probed. Increasing ionic strength led to a dramati c rise in the poly(Glu,Tyr) [4:1 poly(glutamate:tyrosine)] K-m and had little effect on the ATP K-m or k(cat) in Csk-mediated phosphoryl tra nsfer. This finding allowed the dead-end peptide inhibitor EDNEFTA to be characterized as a linear competitive inhibitor of poly(Glu,Tyr) an d a linear noncompetitive inhibitor of ATP. Taken together with previo us data, the overall kinetic mechanism could now be assigned as random substrate binding, ternary complex. Compared to Mn, Mg was shown to s ustain phosphoryl transfer with a 2.5-fold higher k(cat) but K-m's for ATP and poly(Glu,Tyr) that were some 15-20-fold higher. An elevated A DP K-i and microviscosity effects were most suggestive of a kinetic me chanism with fast ADP release, and the chemical step fully rate-determ ining in the Mg-dependent reaction. Steady-state kinetic analyses of C sk reactions with Co and Ni in addition to Mg and Mn on wild-type and D314E Csk with ATP and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate )] as substrates were performed. The k(cat) thio effects [k(cat)(ATP)/ k(cat)(ATP gamma S)] were inversely correlated with metal thiophilicit y in both wild-type and D314E mutant Csk reactions, although the relat ionship was less pronounced in the latter. These results appear to und erscore the role of gamma-phosphoryl hydrogen bending/salt bridging in the wild-type Csk reaction transition state, which is somewhat pertur bed in the D314E Csk reaction. In the case of the Ni reaction, the k(c at) thio effect was reduced to about 2 in the wild-type and D314E muta nt Csk reactions. Relevance with regard to the degree of nucleophilic attack in the transition state, i.e., associative vs dissociative char acter of phosphoryl transfer, is discussed.