Kinetic Mechanism and Rate-Limiting Steps of Focal Adhesion Kinase-1

Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH2, FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k(cat) (0.052 +/- 0.001 s(-1)), K-MgATP (1.2 +/- 0.1 mu M), K-iMgATP (1.3 +/- 0.2 mu M), KFAK-tide (5.6 +/- 0.4 mu M), and KiFAK-tide (6.1 +/- 1.1 mu M) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the beta gamma-bridge:beta-non-bridge positional oxygen exchange of [gamma-O-18(4)]ATP in the presence of 1 mM [gamma-O-18(4)]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k(x)/k(cat) = 0.14 +/- 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k(cat)-KFAK-tide, while k(cat) and k(cat)/K-MgATP were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on K-cat/K-m for MgATP but not on k(cat)/K-m for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (k(cat)) is rate-limited by both reversible phosphoryl group transfer (k(forward) approximate to 0.2 s(-1) and k(reverse) approximate to 0.04 s(-1)) and a slow step (k(conf) approximate to 0.1 s(-1)) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.