Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II

Quantum and molecular mechanics (QM/MM) and QM-only (cluster model) modeling techniques represent the two workhorses in mechanistic understanding of enzyme catalysis. One of the stringent tests for QM/MM and/or QM approaches is to provide quantitative answers to real-world biochemical questions, such as the effect of single-point mutations on enzyme kinetics. This translates into predicting the relative activation energies to 1–2 kcal·mol–1 accuracy; such predictions can be used for the rational design of novel enzyme variants with desired/improved characteristics. Herein, we employ glutamate carboxypeptidase II (GCPII), a dizinc metallopeptidase, also known as the prostate specific membrane antigen, as a model system. The structure and activity of this major cancer antigen have been thoroughly studied, both experimentally and computationally, which makes it an ideal model system for method development. Its reaction mechanism is quite well understood: the reaction coordinate comprises a “tetrahedral intermediate” and two transition states and experimental activation Gibbs free energy of ∼17.5 kcal·mol–1 can be inferred for the known kcat ≈ 1 s–1. We correlate experimental kinetic data (including the E424H variant, newly characterized in this work) for various GCPII mutants (kcat = 8.6 × 10–5 s–1 to 2.7 s–1) with the energy profiles calculated by QM/MM and QM-only (cluster model) approaches. We show that the near-quantitative agreement between the experimental values and the calculated activation energies (ΔH⧧) can be obtained and recommend the combination of the two protocols: QM/MM optimized structures and cluster model (QM) energetics. The trend in relative activation energies is mostly independent of the QM method (DFT functional) used. Last but not least, a satisfactory correlation between experimental and theoretical data allows us to provide qualitative and fairly simple explanations of the observed kinetic effects which are thus based on a rigorous footing.

量子力学与分子力学联用（QM/MM）以及纯量子力学（团簇模型）建模技术，是阐释酶催化反应机制的两大核心工具。对QM/MM乃至纯QM方法的严苛验证标准之一，便是为实际生化问题提供定量解答，例如单点突变对酶促反应动力学的影响。这意味着需将相对活化能的预测精度控制在1~2 kcal·mol⁻¹以内；这类预测可用于理性设计具备预期或优化后特性的新型酶变体。本研究选取双锌金属肽酶谷氨酸羧肽酶II（GCPII，又称前列腺特异性膜抗原）作为模型体系。该蛋白作为重要的癌症抗原，其结构与活性已通过实验与计算手段得到充分研究，因此是方法开发的理想模型体系。其反应机制已得到较为清晰的阐释：反应坐标包含一个“四面体中间体”与两个过渡态；结合已知的催化常数（kcat）≈1 s⁻¹，可推导出实验活化吉布斯自由能约为17.5 kcal·mol⁻¹。本研究将不同GCPII突变体（催化常数kcat范围为8.6×10⁻⁵ s⁻¹至2.7 s⁻¹，其中E424H变体为本工作中新表征得到的）的实验动力学数据，与通过QM/MM及纯QM（团簇模型）方法计算得到的能量剖面进行关联分析。研究结果表明，实验数据与计算得到的活化焓（ΔH⧧）可实现近乎定量的吻合，因此我们推荐结合两种计算方案：采用QM/MM优化结构，结合团簇模型（QM）计算能量学参数。相对活化能的变化趋势基本不受所选用的QM方法（密度泛函理论（DFT）泛函）的影响。最后，实验与理论数据间的良好关联，使我们能够基于严谨的理论依据，对观测到的动力学效应给出定性且简洁易懂的解释。