Kinetic mechanisms of the forward and reverse pp60(c-src) tyrosine kinase reactions

The kinetic mechanism of the pp60(c-src) tyrosine kinase (src TK) reaction was investigated in the forward and reverse directions. In the forward direction, initial velocities obtained by varying ATP and the peptide (FGE)(3)Y(GEF)(2)GD indicated a sequential addition of the two substrates. The peptide analog, (FGE)(3)F(GEF)(2)GD, was a competitive inhibitor versus the peptide substrate and a noncompetitive inhibitor versus MgATP. Interestingly, the tyrosine hydroxyl group imparts only a 6-fold increase in binding. AMP-PCP was a competitive inhibitor versus MgATP and a noncompetitive inhibitor versus the peptide substrate. These results prove that the addition of substrates is random. Furthermore, there appears to be little binding synergy as the K-iMgATP congruent to 2.4K(mMgATP). The phosphorylated peptide (FGE)(3)-pY-(GEF)(2)GD was a competitive inhibitor versus peptide and a noncompetitive inhibitor against MgATP, suggesting that a dead end complex can form between MgATP, the phosphorylated peptide product, and the enzyme. The reverse reaction was investigated by varying ADP and the phosphopeptide, (FGE)(3)-pY-(CEF)(2)GD. The initial velocity pattern was indicative of a sequential mechanism. There was even less binding synergy in the reverse direction as the K-iMgADP congruent to 14K(mMgADP). AMP-CP was a competitive inhibitor versus MgADP and a noncompetitive inhibitor versus the phosphopeptide, (FGE)(3)F(GEF)(2)GD was a competitive inhibitor versus the phosphopeptide and a noncompetitive inhibitor versus MgADP. These data prove that addition of the substrates in the reverse direction is random. (FGE)(3)Y(GEF)(2)GD was a competitive inhibitor against peptide substrate and a noncompetitive inhibitor against MSADP; therefore a dead end complex can form between MgADP, (FGE)(3)Y(GEF)(2)GD, and the enzyme. These results indicate that the src TK reaction follows a sequential bi-bi rapid equilibrium random mechanism in both directions, with dead end complexes forming when either MgATP and (FGE)(3)-pY-(GEF)(2)GD or MgADP and (FGE)(3)Y(GEF)(2)GD bind to the enzyme. The kinetic constants determined from the forward and reverse reactions were used in the Haldane equation to determine a K-eq constant for the forward reaction of 10.1, corresponding to a Delta G of -1.4 kcal/mol. This further confirms that the O-P bond of phosphotyrosine is similar in energy to that of the gamma-phosphoryl of MgATP.