Dissecting substrate recognition by thrombin using the inactive mutant S195A
Dissecting substrate recognition by thrombin using the inactive mutant S195A
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DOI：
10.1016/s0301-4622(02)00289-2
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发表时间：
2003-01-01
影响因子：
3.8
通讯作者：
Di Cera, E
中科院分区：
文献类型：
--
作者：
Krem, MM;Di Cera, E
用无催化活性的突变体S195 A研究了平衡条件下凝血酶与底物的相互作用。通过监测内源荧光的变化，我们测量了各种合成底物，PAR肽和抑制剂PPACK的解离常数。S195 A突变体保留了野生型的Na+结合特性，Na+的存在增强了底物与突变体的结合。温度依赖性研究允许计算活性位点处底物结合的热力学参数，并显示可忽略的DeltaC（p）。在P1-P3位置携带取代的合成底物的滴定显示与野生型水解中鉴定的特异性层次结构一致的能量学。与PAR肽，它与凝血酶的活性位点和外位点I相互作用的滴定，也显示出与野生型在稳态下获得的结果一致。这些研究结果表明，酶的失活突变体使得有可能解剖与底物结合有关的平衡组分，并补充野生型动力学特性的信息。(C)2002 Elsevier Science B. V.保留所有权利。
The catalytically inactive mutant S195A was used to study the interaction of thrombin with substrates under equilibrium conditions. By monitoring changes in intrinsic fluorescence, we measured dissociation constants for a variety of synthetic substrates, PAR peptides and the inhibitor PPACK. The S195A mutant retains the Na+-binding properties of the wild type, and substrate binding to the mutant is enhanced by the presence of Na+. Temperature dependence studies allowed calculation of the thermodynamic parameters of substrate binding at the active site and showed a negligible DeltaC(p). Titration of synthetic substrates carrying substitutions at the P1-P3 positions revealed energetics consistent with the specificity hierarchy identified in hydrolysis by the wild type. Titration with PAR peptides, which interact with both the active site and exosite I of thrombin, also showed consistency with the results obtained with the wild type at steady state. These findings demonstrate that inactive mutants of enzymes make it possible to dissect the equilibrium components linked to substrate binding and complement information on the kinetic properties of the wild type. (C) 2002 Elsevier Science B.V. All rights reserved.