Kinetic mechanistic studies of Cdk5/p25-catalyzed H1P phosphorylation: metal effect and solvent kinetic isotope effect.
Kinetic mechanistic studies of Cdk5/p25-catalyzed H1P phosphorylation: metal effect and solvent kinetic isotope effect.
复制标题
DOI：
10.1021/bi100244j
复制
发表时间：
2010-06-15
期刊：
影响因子：
2.9
通讯作者：
Stein, Ross L.
中科院分区：
文献类型：
--
作者：
Liu, Min;Girma, Eleni;Glicksman, Marcie A.;Stein, Ross L.
Cdk 5/p25是细胞周期蛋白依赖性Ser/Thr激酶家族的成员，已被确定为阿尔茨海默病相关的主要激酶之一，可促进高磷酸化tau的形成，而高磷酸化tau是神经原纤维缠结的主要成分。我们和其他人一直在开发cdk 5/p25抑制剂作为可能的阿尔茨海默病（AD）的治疗药物。为了支持这些努力，我们研究了金属效应和溶剂动力学同位素效应对cdk 5/p25催化的H1 P（一种组蛋白H-1衍生肽）磷酸化的影响。在这里，我们报告说，第二个Mg 2+除了形成的ATP-Mg复合物是需要结合到cdk 5/p25的催化活性。它通过增加kcat激活cdk 5/p25，并诱导有利于ATP结合的构象变化，但对H1 P肽底物的结合亲和力没有影响。第二个Mg 2+的结合不改变底物的结合顺序。该反应遵循相同的快速平衡随机机制，在存在或不存在的第二个Mg 2+证明了由初始速度分析和底物类似物和产物抑制研究。在第二个Mg 2+存在下，线性质子存量的正常SKIE为2.0 ± 0.1，表明在限速磷酰基转移步骤中存在单个质子转移。pH曲线显示pKa为6.5的残基最有可能是促进质子转移的一般酸碱催化剂。
Cdk5/p25 is a member of the cyclin-dependent, Ser/Thr kinase family and has been identified as one of the principle Alzheimer’s disease-associated kinases which promote the formation of hyperphoshorylated tau, the major component of neurofibrillary tangles. We and others have been developing inhibitors of cdk5/p25 as possible therapeutic agents for Alzheimer’s disease (AD). In support of these efforts, we examine the metal effect and solvent kinetic isotope effect on cdk5/p25-catalyzed H1P (a histone H-1-derived peptide) phosphorylation. Here, we report that a second Mg2+ in addition to the one forming the ATP-Mg complex is required to bind to cdk5/p25 for its catalytic activity. It activates cdk5/p25 by demonstrating an increase in kcat and induces a conformational change that favors ATP binding but has no effect on the binding affinity for the H1P peptide substrate. The binding of the second Mg2+ does not change the binding order of substrates. The reaction follows the same rapid equilibrium random mechanism in the presence or absence of the second Mg2+ as evidenced by initial velocity analysis and substrate analogue and product inhibition studies. A linear proton inventory with a normal SKIE of 2.0 ± 0.1 in the presence of the second Mg2+ was revealed and suggested a single proton transfer in the rate-limiting phosphoryl transfer step. pH-profile revealed a residue with a pKa of 6.5 that is most likely the general acid-base catalyst facilitating the proton transfer.
登录
查看更多内容
影响因子：
64.8
作者：
TSAI, LH;DELALLE, I;HARLOW, E
通讯作者：
HARLOW, E
影响因子：
2.9
作者：
Saylor, P;Wang, CQ;Adams, JA
通讯作者：
Adams, JA
影响因子：
2.9
作者：
Adams, JA
通讯作者：
Adams, JA
影响因子：
2.5
作者：
Ishiguro, K;Sato, K;Imahori, K
通讯作者：
Imahori, K
影响因子：
3.5
作者：
BAUMANN, K;MANDELKOW, EM;MANDELKOW, E
通讯作者：
MANDELKOW, E