Posner I, Engel M, Levitzki A
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
J Biol Chem. 1992 Oct 15;267(29):20638-47.
The tyrosine kinase activity of the epidermal growth factor receptor (EGFR-TK) was determined at varying poly-Glu6Ala3Tyr1 (GAT) or [Val5]-angiotensin II (AT) and constant ATP concentrations and vice versa. With GAT as substrate, double reciprocal plots intersected practically on the abscissa following EGFR-TK pre-activation with EGF, but below the abscissa without EGF pre-activation. The EGFR-TK inhibitors App(NH)p (5'-adenylyl-beta, gamma-imidodiphosphate) and ADP were competitive with ATP and noncompetitive with GAT. Four families of 1/v vs. 1/[ATP] plots, constructed at different fixed concentrations of ADP and a different constant concentration of GAT for each family, yielded Slope1/ATP replots which intersected to the left of the ordinate and below the abscissa. GAT and AT, as cosubstrates, were competitive with each other and noncompetitive with ATP; 1/v vs. 1/[GAT] or 1/[AT] plots were hyperbolic and reached horizontal asymptotes when v was expressed as the rate of common product formation. All data were subjected to computer best-fit analysis by a program written especially for this purpose. We conclude that (i) the EGFR-TK reaction follows a Sequential Bi-Bi Rapid Equilibrium Random mechanism, and (ii) EGF induces conformational changes in the EGFR-TK active center which lead to marked decreases in the apparent dissociation constants of both substrates of the kinase reaction and a concomitant increase in initial velocities and Vmax (apparent).
在不同的聚谷氨酸6丙氨酸3酪氨酸（GAT）或[缬氨酸5] -血管紧张素II（AT）浓度以及恒定的ATP浓度下测定表皮生长因子受体（EGFR - TK）的酪氨酸激酶活性，反之亦然。以GAT为底物时，在表皮生长因子（EGF）预激活EGFR - TK后，双倒数图实际上在横坐标上相交，但在没有EGF预激活时则在横坐标下方相交。EGFR - TK抑制剂App(NH)p（5'-腺苷 - β,γ-亚氨二磷酸）和ADP与ATP竞争，与GAT非竞争。在不同的固定ADP浓度下构建了四个1/v对1/[ATP]的图族，每个图族中GAT浓度恒定且不同，得到的斜率1/ATP重绘图在纵坐标左侧和横坐标下方相交。GAT和AT作为共底物，彼此竞争且与ATP非竞争；当v表示为共同产物形成速率时，1/v对1/[GAT]或1/[AT]的图呈双曲线并达到水平渐近线。所有数据均通过专门为此编写的程序进行计算机最佳拟合分析。我们得出结论：（i）EGFR - TK反应遵循顺序双底物双产物快速平衡随机机制，以及（ii）EGF诱导EGFR - TK活性中心的构象变化，这导致激酶反应的两种底物的表观解离常数显著降低，并伴随初始速度和Vmax（表观）增加。
