Generation of nucleophilic character in the Cys25/His159 ion pair of papain involves Trp177 but not Asp158
Generation of nucleophilic character in the Cys25/His159 ion pair of papain involves Trp177 but not Asp158
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DOI：
10.1021/bi702126p
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发表时间：
2008-02-19
期刊：
影响因子：
2.9
通讯作者：
Brocklehurst, Keith
中科院分区：
文献类型：
--
作者：
Gul, Sheraz;Hussain, Syeed;Brocklehurst, Keith
木瓜蛋白酶（EC 3.4.22.2）是半胱氨酸蛋白酶超家族中研究最深入的成员，它的研究极大地促进了我们对非共价相互作用在酶活性中心化学中的作用的理解。以前，我们报道的证据表明，长期以来认为催化能力与催化位点(Cys25)- s -/(His159)- im (+)H离子对的形成同步发展的观点是不正确的，并且每个伙伴都需要构象重排才能发挥其催化作用。非催化“亲密”离子对的伙伴相互溶剂化水平的降低应该释放(Cys25)- s -的亲核特性，并允许(His159)- im +H与底物的离去基结合，以提供其一般的酸催化消除。可以实现这一目标的假设包括离子对的静电调制和Trp177对其疏水屏蔽溶剂的扰动。离催化位点最近的潜在静电调节剂是Asp158，其对Ala的突变大大降低了催化活性。在这里，我们报告了对这些假设的调查，通过计算机建模和停止流动的ph依赖动力学研究相结合，使用一系列新的阳离子氨基烷基2-吡啶二硫化物时间依赖性抑制剂作为反应性探针。这些探针2-4 （n = 2-4）分别以H3N+-[CH2](n)- s - s -2-吡啶基（+）H和H3N+-[CH2](n)- s - s -2-吡啶基（+）H的平衡混合物形式存在，分别在酸性和弱碱性介质中占主导地位。通过建模和动力学分析表明，这些探针2-4 （n = 2-4）在Asp158附近有不同程度的结合效果，在某些情况下也在Trp177附近。通过对2-4和CH3-[CH2](2)- s - s -2-pyridyl(+)H可逆箭头反应的动力学分析和正态模式计算得出结论：Asp158不参与离子对中亲核性质的产生，而对Trp177起关键作用。
Studies on papain (EC 3.4.22.2), the most thoroughly investigated member of the cysteine proteinase superfamily, have contributed substantially to our understanding of the roles of noncovalent interactions in enzyme active center chemistry. Previously, we reported evidence that the long-held view that catalytic competence develops synchronously with formation of the catalytic site (Cys25)-S-/(His159)-Im(+)H ion pair is incorrect and that conformational rearrangement is necessary for each of the partners to play its role in catalysis. A decrease in the level of mutual solvation of the partners of the noncatalytic "intimate" ion pair should release the nucleophilic character of (Cys25)-S- and allow association of (His159)-Im+H with the leaving group of a substrate to provide its general acid-catalyzed elimination. Hypotheses by which this could be achieved involve electrostatic modulation of the ion pair and perturbation of its hydrophobic shielding from solvent by Trp177. The potential electrostatic modulator closest to the catalytic site is Asp158, the mutation of which to Ala substantially decreases catalytic activity. Here we report an investigation of these hypotheses by a combination of computer modeling and stopped-flow pH-dependent kinetic studies using a new series of cationic aminoalkyl 2-pyridyl disulfide time-dependent inhibitors as reactivity probes. These probes 2-4 (n = 2-4), which exist as equilibrium mixtures of H3N+-[CH2](n)-S-S-2-pyridyl(+)H and H3N+-[CH2](n)-S-S-2-pyridyl which predominate in acidic and weakly alkaline media, respectively, were shown by modeling and kinetic analysis to bind with various degrees of effectiveness near Asp158 and in some cases also near Trp177. Kinetic analysis of the reactions of 2-4 and of the reaction of CH3-[CH2](2)-S-S-2-pyridyl(+)H reversible arrow CH3-[CH2](2)-S-S-2-pyridyl 1 and normal mode calculations lead to the conclusion that Asp158 is not involved in the generation of nucleophilic character in the ion pair and demonstrates a key role for Trp177.