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

0552067 - ÚOCHB 2023 RIV US eng J - Journal Article
Bím, Daniel - Navrátil, Michal - Gutten, Ondrej - Konvalinka, Jan - Kutil, Zsofia - Culka, Martin - Navrátil, Václav - Alexandrova, A. N. - Bařinka, Cyril - Rulíšek, Lubomír
Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II.
Journal of Physical Chemistry B. Roč. 126, č. 1 (2022), s. 132-143. ISSN 1520-6106. E-ISSN 1520-5207
R&D Projects: GA MŠMT(CZ) LTAUSA19148; GA ČR(CZ) GA18-14167S; GA ČR(CZ) GJ19-22269Y
Institutional support: RVO:61388963 ; RVO:86652036
Keywords : monoamine-oxidase B * reaction mechanism * membrane antigen
OECD category: Physical chemistry
Impact factor: 3.3, year: 2022
Method of publishing: Limited access
https://doi.org/10.1021/acs.jpcb.1c09240

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.
Permanent Link: http://hdl.handle.net/11104/0327224