Wang Qunzhao, Cahill Sean M, Blumenstein Michael, Lawrence David S
Department of Biochemistry, The Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
J Am Chem Soc. 2006 Feb 15;128(6):1808-9. doi: 10.1021/ja0577692.
A new mechanistic principle by which protein tyrosine kinase substrates fluorescently report the introduction of a phosphate moiety has been developed. NMR was used to establish that tyrosine phosphorylation induces the disruption of pi-pi stacking interactions of the tyrosine moiety with a proximal fluorophore on the peptide substrate. We have demonstrated that (1) the peptide substrates described in this study are useful for a wide variety of different tyrosine kinases, (2) physiological concentrations of ATP can be employed (unlike the standard radioactive ATP kinase assays), thus providing a more realistic assessment of inhibitor potency, and (3) protein kinase self-activation can be observed in real-time.
一种新的机制原理已被开发出来，通过该原理蛋白质酪氨酸激酶底物可通过荧光报告磷酸基团的引入。利用核磁共振确定酪氨酸磷酸化会诱导酪氨酸部分与肽底物上近端荧光团的π-π堆积相互作用的破坏。我们已经证明：（1）本研究中描述的肽底物可用于多种不同的酪氨酸激酶；（2）可以采用生理浓度的ATP（与标准放射性ATP激酶测定不同），从而对抑制剂效力进行更实际的评估；（3）可以实时观察到蛋白激酶的自我激活。
