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

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• 作者：Jessica L. Schneck ; Jacques Briand ; Stephanie Chen ; Ruth Lehr ; Patrick McDevitt ; Baoguang Zhao ; Angela Smallwood ; Nestor Concha ; Khyati Oza ; Robert Kirkpatrick ; Kang Yan ; James P. Villa ; Thomas D. Mee
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：August 24, 2010
• 年：2010
• 卷：49
• 期：33
• 页码：7151-7163
• 全文大小：1026K
• 年卷期：v.49,no.33(August 24, 2010)
• ISSN：1520-4995

文摘

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 μM), K_iMgATP (1.3 ± 0.2 μM), K_FAK-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 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 ≈ 0.2 s⁻¹ and k_reverse ≈ 0.04 s⁻¹) and a slow step (k_conf ≈ 0.1 s⁻¹) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.