Laboratory of Structural and Mechanistic Enzymology, School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, UK.
Biochemistry. 2011 Dec 13;50(49):10732-42. doi: 10.1021/bi201207z. Epub 2011 Nov 17.
Understanding the roles of noncovalent interactions within the enzyme molecule and between enzyme and substrate or inhibitor is an essential goal of the investigation of active center chemistry and catalytic mechanism. Studies on members of the papain family of cysteine proteinases, particularly papain (EC 3.4.22.2) itself, continue to contribute to this goal. The historic role of the catalytic site Cys/His ion pair now needs to be understood within the context of multiple dynamic phenomena. Movement of Trp177 may be necessary to expose His159 to solvent with consequent decrease in its degree of electrostatic solvation of (Cys25)-S(-). Here we report an investigation of this possibility using computer modeling of quasi-transition states and pH-dependent kinetics using 3,3'-dipyridazinyl disulfide, its n-propyl and phenyl derivatives, and 4,4'-dipyrimidyl disulfide as reactivity probes that differ in the location of potential hydrogen-bonding acceptor atoms. Those interactions that influence ion pair geometry and thereby catalytic competence, including by transmission of the modulatory effect of a remote ionization with pK(a) 4, were identified. A key result is the correlation between the kinetic influence of the modulatory trigger of pK(a) 4 and disruption of the hydrogen bond donated by the indole N-H of Trp177, the hydrophobic shield of the initial "intimate" ion pair. This hydrogen bond is accepted by the amide O of Gln19-a component of the oxyanion hole that binds the tetrahedral species formed from the substrate during the catalytic act. The disruption would be expected to contribute to the mobility of Trp177 and possibly to the effectiveness of the binding of the developing oxyanion.
理解非共价相互作用在酶分子内以及酶与底物或抑制剂之间的作用是研究活性中心化学和催化机制的重要目标。木瓜蛋白酶家族的成员，特别是木瓜蛋白酶（EC 3.4.22.2）本身的研究继续为这一目标做出贡献。催化部位 Cys/His 离子对的历史作用现在需要在多种动态现象的背景下加以理解。Trp177 的移动可能是必需的，以使 His159暴露于溶剂中，从而降低（Cys25）-S（-）的静电溶剂化程度。在这里，我们使用准过渡态的计算机建模和 pH 依赖性动力学研究了这种可能性，使用 3,3'-二吡啶二嗪基二硫化物、其正丙基和苯基衍生物以及 4,4'-二嘧啶基二硫化物作为反应性探针，这些探针在潜在氢键接受原子的位置上有所不同。那些影响离子对几何形状从而影响催化能力的相互作用，包括通过远程离解的调制效应的传递，离解常数为 pK(a) 4，都被识别出来。一个关键的结果是，pK(a) 4 的调制触发的动力学影响与破坏 Trp177 的吲哚 N-H 捐赠的氢键之间存在相关性，这种氢键被 Gln19 的酰胺 O 接受，Gln19 是与催化过程中形成的底物的四面体物种结合的氧阴离子空穴的组成部分。这种氢键的破坏预计会导致 Trp177 的移动性增强，并可能导致正在形成的氧阴离子的结合更有效。
