Probing Proton Bridges in Catalysis by Thrombin

描述（由申请人提供）： 该提案的主要目标是通过高分辨率，低场1H NMR表征，在人凝血酶的催化过程中动员的质子桥，主要是在活性位点，但也在子位点。据推测，短，强氢键（SSHB）形成在活性位点时，催化的His得到质子化的凝血酶在酸滴定，或由于与高选择性抑制剂的相互作用。这些类似于底物水解催化过程中的过渡态（TS）事件。本研究中的大多数凝血酶抑制剂都是抗凝血作用药物设计的目标。SSHB被认为是酶用于降低TS自由能的有效的酸碱催化模式。对凝血酶的变构功能至关重要的其他特定位点也很可能具有SSHB。SSHB的特征在于核间氢键供体和受体距离< 2.6  在线性氢键中，高度去屏蔽的质子共振信号，低于1的分馏因子和快速的交换速率。将根据1H NMR化学位移和分馏因子计算键长。将在存在和不存在Na+离子的情况下分别研究SSHB：1）凝血酶在Tris缓冲液中的pH滴定; 2）酰化中的TS模拟物，a）D-Phe-Pro-ArgCH 2Cl抑制的，B）肽基吡啶鎓甲基酮抑制的，在活性位点和亚位点都有相互作用，和c）与凝血酶的酮-酰胺过渡态模拟复合物; 3）酰化酶的模拟物4）模拟TS进行脱酰的凝血酶的三种类型的膦酸酯共价加合物。5)水蛭素抑制凝血酶与亚位点，特别是阴离子外位点的相互作用。水蛭素及其类似物在粘原存在下和在重水中与凝血酶的结合将补充NMR研究。这些研究的实际意义在于为设计基于机制或过渡态类似物的凝血酶抑制剂提供指导。这对于理解酶的催化能力和酶的进化理论也具有重要意义。

DESCRIPTION (provided by applicant): The main goal of this proposal is to characterize by high-resolution, low-field 1H NMR, the proton bridges mobilized in the catalytic process in human thrombin, predominantly at the active site but also at subsites. It is hypothesized that short, strong hydrogen bonds (SSHBs) form at the active site when the catalytic His gets protonated in thrombin as in acid titration, or due to interactions with highly selective inhibitors. These are analogous to events at the transitions states (TS) in the course of catalysis of substrate hydrolysis. Most thrombin inhibitors in this study are targets of drug design for anticoagulant effects. SSHBs are believed to be an effective mode of acid-base catalysis employed by enzymes to reduce the TS free energy. Other specific sites critical for the allosteric function of thrombin are very likely to also have SSHBs. SSHBs will be characterized by internuclear hydrogen bond donor and acceptor distances < 2.6  in a linear H-bond, highly deshielded proton resonance signals, fractionation factors below unity and rapid exchange rates. The bond lengths will be calculated from both 1H NMR chemical shifts and fractionation factors. SSHBs will be studied each in the presence and absence of Na+ ions: in 1) pH titrations of thrombin in Tris buffer; 2) TS mimics in acylation, a) D-Phe-Pro-ArgCH2CI-inhibited, b) a peptidyl pyridinium methyl ketone-inhibited, with interactions at both active sites and subsites and c) a keto-amide transition state mimic complex with thrombin; 3) a mimic of the acylenzyme 4) three types of phosphonate ester covalent adducts of thrombin mimicking the TS for deacylation. 5) hirudin-inhibited thrombin with interactions at subsites and particularly at the anionic exosite. Binding of hirudin and its analogs to thrombin in the presence of viscogens and in heavy water will complement the NMR studies. A practical significance of these studies is the provision of guidelines for the design of mechanism-based or transition-state-analog inhibitors of thrombin. A great significance is also to basic understanding of enzyme catalytic power and enzyme-evolutionary theory.