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首页> 外文期刊>Biochemistry >Kinetic Mechanistic Studies of Cdk5/p25-Catalyzed HIP Phosphorylation: Metal Effect and Solvent Kinetic Isotope Effect
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Kinetic Mechanistic Studies of Cdk5/p25-Catalyzed HIP Phosphorylation: Metal Effect and Solvent Kinetic Isotope Effect

机译：CDK5 / P25催化髋关节磷酸化的动力学研究：金属效应和溶剂动力学同位素效应

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摘要

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 that promote the formation of hyperphosphorylated 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 HIP (a histone H-1-derived peptide) phosphorylation. Here, we report that a second Mg2+ in addition to the one forming the MgATP complex is required to bind to cdk5/p25 for its catalytic activity. It activates cdk5/p25 by demonstrating an increase in kcet and induces a conformational change that favors ATP binding but has no effect on the binding affinity for the HIP 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. The pH profile revealed a residue with a pK a of 6.5 that is most likely the general acid—base catalyst facilitating the proton transfer.
机译：CDK5 / 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Alzheimer的疾病相关激酶原则之一，这些激酶促进了多磷酸化Tau的形成，神经纤维缠结的主要成分。我们和其他人一直在为阿尔茨海默病（AD）的可能治疗剂的CDK5 / P25的抑制剂。为了支持这些努力，我们研究了对CDK5 / P25催化髋关节（组蛋白H-1-衍生的肽）磷酸化的金属效果和溶剂动力学同位素效应。这里，我们报告说，除了形成MgATP复合物的一个外，还需要第二Mg2 +，以结合其催化活性的CDK5 / P25。它通过演示KCET增加来激活CDK5 / P25，并诱导有利于ATP结合但对髋肽底物的结合亲和力产生影响的构象变化。第二Mg2 +的结合不会改变基材的结合顺序。在存在或不存在第二Mg2 +的情况下，反应遵循相同的快速平衡随机机制，如初始速度分析和衬底类似物和产品抑制研究所证明的。揭示了在第二Mg2 +存在下具有2.0±0.1的正常滑块的线性质子库存，并提出了速率限制磷酸转移步骤中的单个质子转移。 pH型谱显示残留物，PK A为6.5，最有可能促进质子转移的一般酸碱催化剂。

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  • 来源
    《Biochemistry》 |2010年第23期|共9页
  • 作者

    Min Liu; Eleni Girma; Marcie A. Glicksman; Ross L. Stein;

  • 作者单位

    Laboratory for Drug Discovery in Neurodegeneration Harvard NeuroDiscovery Center 65 Landsdowne Street Fourth Floor Cambridge Massachusetts 02139;

    Laboratory for Drug Discovery in Neurodegeneration Harvard NeuroDiscovery Center 65 Landsdowne Street Fourth Floor Cambridge Massachusetts 02139;

    Laboratory for Drug Discovery in Neurodegeneration Harvard NeuroDiscovery Center 65 Landsdowne Street Fourth Floor Cambridge Massachusetts 02139;

    Sirtris Pharmaceuticals Cambridge Massachusetts 02139;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    member; formation; effect;

    机译：会员;形成;效果;

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