Divalent ion effects and insights into the catalytic mechanism of protein tyrosine kinase Csk

Miranda R. Grace, Christopher T. Walsh, Philip A. Cole

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Abstract

Csk (C-terminal Src kinase) is a protein tyrosine kinase which catalyzes the transfer of the γ-phosphoryl group of ATP to the tyrosine hydroxyl of proteins in the presence of a divalent ion. Previous work with poly(Glu,Tyr) as the tyrosine-containing substrate and Mn as the divalent ion defined a ternary complex mechanism with ADP product release partially rate-determining [Cole, P. A., et al. (1994) J. Biol. Chem. 269, 30880-30887]. In this current study, ionic strength and divalent ion effects were probed. Increasing ionic strength led to a dramatic 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 transfer. This finding allowed the dead- end peptide inhibitor EDNEFTA to be characterized as a linear competitive inhibitor of poly(Glu,Tyr) and a linear noncompetitive inhibitor of ATP. Taken together with previous data, the overall kinetic mechanism could now be assigned as random substrate binding, ternary complex. Compared to Mn, Mg was shown to sustain 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 ADP K(i) and microviscosity effects were most suggestive of a kinetic mechanism with fast ADP release, and the chemical step fully rate-determining in the Mg-dependent reaction. Steady-state kinetic analyses of Csk reactions with Co and Ni in addition to Mg and Mn on wild-type and D314E Csk with ATP and ATPγS [adenosine 5'-O-(3-thiotriphosphate)] as substrates were performed. The k(cat) thio effects [k(cat)(ATP)/k(cat)(ATPγS)] were inversely correlated with metal thiophilicity in both wild-type and D314E mutant Csk reactions, although the relationship was less pronounced in the latter. These results appear to underscore the role of γ-phosphoryl hydrogen bonding/salt bridging in the wild-type Csk reaction transition state, which is somewhat perturbed in the D314E Csk reaction. In the case of the Ni reaction, the k(cat) thio effect was reduced to about 2 in the wild-type and D314E mutant Csk reactions. Relevance with regard to the degree of nucleophilic attack in the transition state, i.e., associative vs dissociative character of phosphoryl transfer, is discussed.

Original languageEnglish (US)
Pages (from-to)1874-1881
Number of pages8
JournalBiochemistry
Volume36
Issue number7
DOIs
StatePublished - Feb 18 1997

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ASJC Scopus subject areas

  • Biochemistry

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