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-NH(2), 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 microM), K(iMgATP) (1.3 +/- 0.2 microM), K(FAK-tide) (5.6 +/- 0.4 microM), and K(iFAK-tide) (6.1 +/- 1.1 microM) 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 betagamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-(18)O(4)]ATP in the presence of 1 mM [gamma-(18)O(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)/K(FAK-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) approximately 0.2 s(-1) and k(reverse) approximately 0.04 s(-1)) and a slow step (k(conf) approximately 0.1 s(-1)) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.
Schneck
Jessica L
JL
Department of Biological Reagents, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426-0989, USA.
Briand
Jacques
J
Chen
Stephanie
S
Lehr
Ruth
R
McDevitt
Patrick
P
Zhao
Baoguang
B
Smallwood
Angela
A
Concha
Nestor
N
Oza
Khyati
K
Kirkpatrick
Robert
R
Yan
Kang
K
Villa
James P
JP
Meek
Thomas D
TD
Thrall
Sara H
SH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D051417:Focal Adhesion Kinase 1 / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding
D010455:Peptides; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D051417:Focal Adhesion Kinase 1