Kinetic mechanism and rate-limiting steps of focal adhesion kinase-1

Abstract

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 kcat (0.052 ± 0.001 s-1), KMgATP (1.2 ± 0.1 μM), KiMgATP (1.3 ± 0.2 μM), KFAK-tide (5.6 ± 0.4 μM), and K iFAK-tide (6.1 ± 1.1 μM) 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 βγ-bridge:β-nonbridge positional oxygen exchange of [γ-18O4]ATP in the presence of 1 mM [γ- 18O4]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 kx/kcat = 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 kcat/KFAK-tide, while kcat and kcat/KMgATP 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 kcat/Km for MgATP but not on kcat/K m for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (kcat) is rate-limited by both reversible phosphoryl group transfer (kforward ≈ 0.2 s-1 and kreverse ≈ 0.04 s-1) and a slow step (kconf ≈ 0.1 s-1) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release. © 2010 American Chemical Society.