Angeles T S, Yang S X, Steffler C, Dionne C A
Cephalon, Inc., West Chester, Pennsylvania 19380, USA.
Arch Biochem Biophys. 1998 Jan 15;349(2):267-74. doi: 10.1006/abbi.1997.0490.
The kinetic mechanism of the trk receptor-linked tyrosine kinase was determined using a baculovirus expressed trk kinase domain and a bacterially expressed phospholipase C-gamma/glutathione S-transferase (PLC-gamma/ GST) fusion protein as substrate. Product and dead-end inhibition studies indicate an ordered association of substrates to trkA kinase with the nucleotide ATP binding prior to the exogenous substrate PLC-gamma/GST, followed by release of the phosphorylated PLC-gamma/GST product prior to release of ADP (sequential ordered bi-bi mechanism). This is in contrast to the reported kinetic mechanisms of closely related EGF receptor and insulin receptor kinases which appear to proceed via a rapid equilibrium random mechanism. The indolocarbazole K-252a, which was previously shown to be a potent and relatively selective inhibitor of trk kinase activity, acts as a competitive inhibitor with respect to ATP. The data suggest that potent and selective kinase inhibitors can be rationally designed by exploring subtle variations surrounding the nucleotide binding sites of receptor tyrosine kinases.
利用杆状病毒表达的trk激酶结构域和细菌表达的磷脂酶C-γ/谷胱甘肽S-转移酶（PLC-γ/GST）融合蛋白作为底物，确定了trk受体相关酪氨酸激酶的动力学机制。产物和终产物抑制研究表明，底物与trkA激酶按顺序结合，核苷酸ATP先于外源底物PLC-γ/GST结合，随后在ADP释放之前释放磷酸化的PLC-γ/GST产物（顺序有序双底物机制）。这与报道的密切相关的表皮生长因子受体和胰岛素受体激酶的动力学机制相反，后者似乎通过快速平衡随机机制进行。吲哚咔唑K-252a先前被证明是trk激酶活性的有效且相对选择性的抑制剂，它对ATP起竞争性抑制剂的作用。数据表明，通过探索受体酪氨酸激酶核苷酸结合位点周围的细微差异，可以合理设计出有效的选择性激酶抑制剂。
