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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F22%3A00552067" target="_blank" >RIV/61388963:_____/22:00552067 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/86652036:_____/22:00552067 RIV/00216208:11310/22:10455451

Výsledek na webu

<a href="https://doi.org/10.1021/acs.jpcb.1c09240" target="_blank" >https://doi.org/10.1021/acs.jpcb.1c09240</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/acs.jpcb.1c09240" target="_blank" >10.1021/acs.jpcb.1c09240</a>

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10403 - Physical chemistry

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Physical Chemistry B

ISSN

1520-6106

e-ISSN

1520-5207

Svazek periodika

126

Číslo periodika v rámci svazku

1

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

12

Strana od-do

132-143

Kód UT WoS článku

000740501700001

EID výsledku v databázi Scopus

2-s2.0-85122825932