Evaluation of the catalytic mechanism of recombinant human Csk (C-terminal Src kinase) using nucleotide analogs and viscosity effects

Philip A. Cole, Paul Burn, Bêla Takacs, Christopher T. Walsh

Research output: Contribution to journalArticlepeer-review

Abstract

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 K(m) values that were in reasonable agreement with literature values reported for the non- recombinant enzyme and with k(cat) about 40 min-1. A sequential kinetic mechanism is suggested by a steady state kinetic analysis. Inhibitor studies with ADP and β,γ-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 k(cat) about 15-20-fold lower than the ATP reaction but with similar K(m) values. Deuterium solvent isotope effects on k(cat) 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.

Original languageEnglish (US)
Pages (from-to)30880-30887
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number49
StatePublished - Dec 9 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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