QM/MM Simulation (B3LYP) of the RNase A Cleavage-Transesterification Reaction Supports a Triester A_N + D_N Associative Mechanism with an O2′ H Internal Proton Transfer

The mechanism of the backbone cleavage-transesterification step of the RNase A enzyme remains controversial even after 60 years of study. We report quantum mechanics/molecule mechanics (QM/MM) free energy calculations for two optimized reaction paths based on an analysis of all structural data and identified by a search for reaction coordinates using a reliable quantum chemistry method (B3LYP), equilibrated structural optimizations, and free energy estimations. Both paths are initiated by nucleophilic attack of the ribose O2′ oxygen on the neighboring diester phosphate bond, and both reach the same product state (PS) (a O3′–O2′ cyclic phosphate and a O5′ hydroxyl terminated fragment). Path 1, resembles the widely accepted dianionic transition-state (TS) general acid (His119)/base (His12) classical mechanism. However, this path has a barrier (25 kcal/mol) higher than that of the rate-limiting hydrolysis step and a very loose TS. In Path 2, the proton initially coordinating the O2′ migrates to the nonbridging O1P in the initial reaction path rather than directly to the general base resulting in a triester (substrate as base) AN + DN mechanism with a monoanionic weakly stable intermediate. The structures in the transition region are associative with low barriers (TS1 10, TS2 7.5 kcal/mol). The Path 2 mechanism is consistent with the many results from enzyme and buffer catalyzed and uncatalyzed analog reactions and leads to a PS consistent with the reactive state for the following hydrolysis step. The differences between the consistently estimated barriers in Path 1 and 2 lead to a 1011 difference in rate strongly supporting the less accepted triester mechanism.即便历经60余年研究，核糖核酸酶A（RNase A）的主链裂解-转酯化步骤机制仍存在争议。本研究基于对所有结构数据的分析，结合可靠量子化学方法（B3LYP）对反应坐标的搜索、平衡结构优化及自由能估算，针对两条优化后的反应路径开展了量子力学/分子力学（QM/MM）自由能计算。两条路径均以核糖O2'氧原子对邻近二酯磷酸键的亲核进攻为起始步骤，最终均达到同一产物态（PS），即O3'–O2'环磷酸酯与O5'羟基封端片段。路径1契合被广泛认可的双阴离子过渡态（TS）广义酸（His119）/碱（His12）经典机制，但该路径的能垒（25 kcal/mol）高于限速水解步骤的能垒，且过渡态结构极为松散。在路径2中，最初配位O2'的质子迁移至非桥连O1P位点，而非直接转移至广义碱，由此形成以底物为碱的三酯AN+DN机制，并伴随单阴离子弱稳定中间体。过渡区域的结构为缔合型，能垒较低（TS1为10 kcal/mol，TS2为7.5 kcal/mol）。路径2机制与酶催化、缓冲液催化及无催化的类似反应所得诸多结果相符，且其产物态与后续水解步骤的反应态一致。路径1与路径2中经一致估算得到的能垒差异，可导致反应速率产生10^11量级的差距，这有力支持了此前未被广泛接受的三酯机制。