Kots Ekaterina D, Khrenova Maria G, Lushchekina Sofya V, Varfolomeev Sergei D, Grigorenko Bella L, Nemukhin Alexander V
Chemistry Department, M.V. Lomonosov Moscow State University , Leninskie Gory 1/3, Moscow 119991, Russian Federation.
N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences , Kosygina 4, Moscow 119334, Russian Federation.
J Phys Chem B. 2016 May 12;120(18):4221-31. doi: 10.1021/acs.jpcb.6b02542. Epub 2016 Apr 29.
The complete catalytic cycle of aspartoacylase (ASPA), a zinc-dependent enzyme responsible for cleavage of N-acetyl-l-aspartate, is characterized by the methods of molecular modeling. The reaction energy profile connecting the enzyme-substrate (ES) and the enzyme-product (EP) complexes is constructed by the quantum mechanics/molecular mechanics (QM/MM) method assisted by the molecular dynamics (MD) simulations with the QM/MM potentials. Starting from the crystal structure of ASPA complexed with the intermediate analogue, the minimum-energy geometry configurations and the corresponding transition states are located. The stages of substrate binding to the enzyme active site and release of the products are modeled by MD calculations with the replica-exchange umbrella sampling technique. It is shown that the first reaction steps, nucleophilic attack of a zinc-bound nucleophilic water molecule at the carbonyl carbon and the amide bond cleavage, are consistent with the glutamate-assisted mechanism hypothesized for the zinc-dependent hydrolases. The stages of formation of the products, acetate and l-aspartate, and regeneration of the enzyme are characterized for the first time. The constructed free energy diagram from the reactants to the products suggests that the enzyme regeneration, but not the nucleophilic attack of the catalytic water molecule, corresponds to the rate-determining stage of the full catalytic cycle of ASPA.
天冬氨酸酰基转移酶（ASPA）是一种负责裂解N-乙酰-L-天冬氨酸的锌依赖性酶，其完整的催化循环通过分子建模方法进行表征。通过量子力学/分子力学（QM/MM）方法并借助QM/MM势的分子动力学（MD）模拟，构建连接酶-底物（ES）和酶-产物（EP）复合物的反应能量分布。从天冬氨酸酰基转移酶与中间类似物复合的晶体结构出发，确定最低能量几何构型和相应的过渡态。采用副本交换伞形采样技术，通过MD计算对底物与酶活性位点结合以及产物释放的阶段进行建模。结果表明，最初的反应步骤，即锌结合的亲核水分子对羰基碳的亲核攻击和酰胺键裂解，与为锌依赖性水解酶假设的谷氨酸辅助机制一致。首次对产物乙酸盐和L-天冬氨酸的形成阶段以及酶的再生进行了表征。构建的从反应物到产物的自由能图表明，酶的再生而非催化水分子的亲核攻击，对应于天冬氨酸酰基转移酶完整催化循环的速率决定阶段。
