Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation.
Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation.
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通过近膜自磷酸化部分激活胰岛素受体激酶结构域。
DOI：
10.1021/bi9809122
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Kohanski,RA
中科院分区：
文献类型：
--
作者：
Cann,AD;Bishop,SM;Ablooglu,AJ;Kohanski,RA
受体酪氨酸激酶的酶活性增加发生在激活环中的一个或两个酪氨酸的反式磷酸化后，位于催化裂缝附近。胰岛素受体的激酶结构域的部分激活，观察到在稀释浓度的激酶，表明顺式自磷酸化的发生。部分激活期间的自磷酸化映射到质膜（JM）酪氨酸，而不是激活环酪氨酸。此外，一个双JM酪氨酸-苯丙氨酸突变激酶（JMY 2F）没有进行部分激活，但催化底物磷酸化在一个非常低的速率。肽磷酸化的稳态动力学进行了测定与JM自磷酸化。JMY 2F突变体用于防止同时顺式自磷酸化，因此在动力学分析中接近基础状态脱辅基酶。肽底物的米氏常数从基础状态的KM，PEP≥ 2.5 mM下降到部分活化状态的0.2 mM，部分活化占主导地位; KM，AT在0.1 mM时几乎保持不变，kcat从180 min-1增加到600 min-1。高KM，PEP表明肽底物与脱辅基酶的弱结合。对于用作JM自磷酸化抑制剂的肽底物，Ki> 1 mM证实了这一点。在cat和KM，ATP中没有明显的变化，表明JM区主要是反式磷酸化反应中肽进入步骤的强屏障。因此，JM区在胰岛素受体激酶结构域的基础状态下起着乳囊内抑制剂的作用。
Increased enzymatic activity of receptor tyrosine kinases occurs after trans-phosphorylation of one or two tyrosines in the activation loop, located near the catalytic cleft. Partial activation of the insulin receptor's kinase domain was observed at dilute concentrations of kinase, suggesting that cis-autophosphorylation was occurring. Autophosphorylation during partial activation mapped to the juxtamembrane (JM) tyrosines and not to activation loop tyrosines. Furthermore, a double JM Tyr-to-Phe mutant kinase (JMY2F) did not undergo partial activation but catalyzed substrate phosphorylation at a very low rate. Steady-state kinetics of peptide phosphorylation were determined with and without JM autophosphorylation. The JMY2F mutant was used to prevent concurrent cis-autophosphorylation and therefore to approximate the basal state apoenzyme in the kinetic analysis. Partial activation was dominated by a decreased Michaelis constant for peptide substrate, fromKM,PEP≥ 2.5 mM in the basal state to 0.2 mM in the partially activated state; theKM,ATPremained virtually unchanged at ∼1 mM, andkcatincreased from 180 to 600 min-1. The highKM,PEPsuggests weak binding of peptide substrates to the apoenzyme. This was confirmed byKi> 1 mM for peptide substrates used as inhibitors of JM autophosphorylation. The absence of comparably large changes inkcatandKM,ATPsuggests that the JM region is primarily a strong barrier to the peptide entry step of trans-phosphorylation reactions. The JM region therefore functions as an intrasteric inhibitor in the basal state of the insulin receptor's kinase domain.