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) reac tion was investigated in the forward and reverse directions. In the fo rward direction, initial velocities obtained by varying ATP and the pe ptide (FGE)(3)Y(GEF)(2)GD indicated a sequential addition of the two s ubstrates. 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 vers us MgATP and a noncompetitive inhibitor versus the peptide substrate. These results prove that the addition of substrates is random. Further more, there appears to be little binding synergy as the K-iMgATP congr uent to 2.4K(mMgATP). The phosphorylated peptide (FGE)(3)-pY-(GEF)(2)G D was a competitive inhibitor versus peptide and a noncompetitive inhi bitor against MgATP, suggesting that a dead end complex can form betwe en MgATP, the phosphorylated peptide product, and the enzyme. The reve rse 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 rev erse direction as the K-iMgADP congruent to 14K(mMgADP). AMP-CP was a competitive inhibitor versus MgADP and a noncompetitive inhibitor vers us 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 dire ction is random. (FGE)(3)Y(GEF)(2)GD was a competitive inhibitor again st peptide substrate and a noncompetitive inhibitor against MSADP; the refore 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 follo ws a sequential bi-bi rapid equilibrium random mechanism in both direc tions, 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 w ere 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.