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Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II
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SYSNO ASEP 0552067 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Predicting Effects of Site-Directed Mutagenesis on Enzyme Kinetics by QM/MM and QM Calculations: A Case of Glutamate Carboxypeptidase II Author(s) Bím, Daniel (UOCHB-X) ORCID, RID
Navrátil, Michal (UOCHB-X) RID
Gutten, Ondrej (UOCHB-X) RID, ORCID
Konvalinka, Jan (UOCHB-X) RID, ORCID
Kutil, Zsofia (BTO-N) RID, ORCID
Culka, Martin (UOCHB-X) ORCID
Navrátil, Václav (UOCHB-X) RID, ORCID
Alexandrova, A. N. (US)
Bařinka, Cyril (BTO-N) RID, ORCID
Rulíšek, Lubomír (UOCHB-X) RID, ORCIDSource Title Journal of Physical Chemistry B. - : American Chemical Society - ISSN 1520-6106
Roč. 126, č. 1 (2022), s. 132-143Number of pages 12 s. Language eng - English Country US - United States Keywords monoamine-oxidase B ; reaction mechanism ; membrane antigen OECD category Physical chemistry R&D Projects LTAUSA19148 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) GA18-14167S GA ČR - Czech Science Foundation (CSF) GJ19-22269Y GA ČR - Czech Science Foundation (CSF) Method of publishing Limited access Institutional support UOCHB-X - RVO:61388963 ; BTO-N - RVO:86652036 UT WOS 000740501700001 EID SCOPUS 85122825932 DOI 10.1021/acs.jpcb.1c09240 Annotation 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. Workplace Institute of Organic Chemistry and Biochemistry Contact asep@uochb.cas.cz ; Kateřina Šperková, Tel.: 232 002 584 ; Jana Procházková, Tel.: 220 183 418 Year of Publishing 2023 Electronic address https://doi.org/10.1021/acs.jpcb.1c09240
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