Catalytic Mechanism of the ppGalNAcT2 Retaining
Glycosyltransferase Inferred From QM/MM Calculations

TRNKA, Tomáš, Stanislav KOZMON, Igor TVAROŠKA and Jaroslav KOČA. Catalytic Mechanism of the ppGalNAcT2 Retaining Glycosyltransferase Inferred From QM/MM Calculations. In CEITEC Annual Conference. 2014.

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TY  - CONF
ID  - 1230979
AU  - Trnka, Tomáš - Kozmon, Stanislav - Tvaroška, Igor - Koča, Jaroslav
PY  - 2014
TI  - Catalytic Mechanism of the ppGalNAcT2 Retaining Glycosyltransferase Inferred From QM/MM Calculations
KW  - glycosyltransferases;reaction mechanism;quantum chemistry
N2  - To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals.
ER  -

Basic information
Original name	Catalytic Mechanism of the ppGalNAcT2 Retaining Glycosyltransferase Inferred From QM/MM Calculations
Authors	TRNKA, Tomáš, Stanislav KOZMON, Igor TVAROŠKA and Jaroslav KOČA.
Edition	CEITEC Annual Conference, 2014.

Other information
Original language	English
Type of outcome	Presentations at conferences
Field of Study	10403 Physical chemistry
Country of publisher	Czech Republic
Confidentiality degree	is not subject to a state or trade secret
Organization unit	Faculty of Science
Keywords in English	glycosyltransferases;reaction mechanism;quantum chemistry
Changed by	Changed by: Ing. Tomáš Trnka, Ph.D., učo 395845. Changed: 19/3/2015 17:32.

Abstract

To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals.

Links
ED1.1.00/02.0068, research and development project	Name: CEITEC - central european institute of technology
286154, interní kód MU	Name: SYLICA - Synergies of Life and Material Sciences to Create a New Future (Acronym: SYLICA)
286154, interní kód MU	Investor: European Union, Capacities

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Catalytic Mechanism of the ppGalNAcT2 Retaining Glycosyltransferase Inferred From QM/MM Calculations

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