Insight into tyrosine phosphorylation in v-Fps using proton inventory techniques.
The phosphoryl group transfer step in the kinase domain of v-Fps, a nonreceptor tyrosine protein kinase, was analyzed using proton inventory and viscosometric techniques. The latter studies show that two nine-residue peptide substrates for v-Fps, peptides I (EAEAYEAIE) and II (EAEIYEAIE), are in rapid equilibrium with the active site and bind with similar affinities (Ks = 2.2 and 1.7 mM for peptides I and II). While phosphoryl group transfer is the rate-limiting step in kcat for peptide I (5 s-1) at neutral pH, peptide II is converted to product by a kinetic mechanism in which phosphoryl group transfer (45 s-1) and product release (20 s-1) partially control this parameter. Significant solvent isotope effects on kcat (k0/kn approximately 1.6) are observed for the phosphorylation of both peptides in 95% D2O. Proton inventories on kcat for peptide I are linear, indicating that the phosphoryl group transfer step is associated with a single proton transfer. Conversely, proton inventories on kcat for peptide II are "bowed" up, consistent with a "virtual" transition state in which phosphoryl group transfer and product release steps partially control this parameter. The lack of solvent isotope effects on kcat/Kpeptide for both peptides can be explained by an equilibrium isotope effect on substrate binding that offsets the kinetic isotope effect for phosphoryl group transfer. In keeping with this proposal, the KI for the inhibitor peptide, EAEIFEAIE, is 11 +/- 0.80 and 6.5 +/- 0.82 mM in 0 and 60% D2O, respectively. Fitting of the proton inventory plots using modified forms of the Gross-Butler equation provide intrinsic isotope effects of 1.7 and 3.6 for peptides I and II. The combined data are consistent with a mechanism involving either an acid-base catalyst or a conformational change preceding the release of products that is accompanied by the disruption of a single hydrogen donor-acceptor pair.
Adams
J A
JA
Department of Chemistry, San Diego State University, California 92182-1030, USA.
D000595:Amino Acid Sequence; D017666:Deuterium Oxide; D015681:Fusion Proteins, gag-onc / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011522:Protons; D012997:Solvents; D014443:Tyrosine / Q000378:metabolism*; D014783:Viscosity
D015681:Fusion Proteins, gag-onc; D007554:Isotopes; D011522:Protons; D012997:Solvents; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C068415:v-fps oncogene protein, Fujinami sarcoma virus; D017666:Deuterium Oxide