Hybrid Quantum Mechanical/Molecular Mechanical Investigation of the beta-1,4-Galactosyltransferase-I Mechanism

作者：Krupicka Martin; Tvaroska Igor^*

来源：Journal of Physical Chemistry B, 2009, 113(32): 11314-11319.

DOI：10.1021/jp904716t

The enzyme beta-1,4-galactosyltransferase-1 (beta 4Gal-T1) catalyzes the transfer of a galactose residue from UDP-Gal to the C4-hydroxyl group of N-acetylglucosamine. The catalytic mechanism of beta 4Gal-T1 was investigated using the hybrid quantum mechanical/molecular mechanical (QM/MM) method, with the QM portion containing 253 atoms treated with density functional theory (DFT) at the BP/DZP and BP/TZ2P levels. The remaining parts of the beta 4Gal-T1 complex, 4527 atoms in all, were modeled using the AMBER molecular force field. A theoretical model of the Michaelis complex was built using the X-ray structure of beta 4Gal-T1 in a complex with the donor or acceptor substrate, respectively. The hybrid QM(DFT)/MM calculations identified all S(N)2-type transition state for the nucleophilic attack of the O4(a) oxygen on the anomeric carbon C1 and the breaking of the C1-O1 glycosidic linkage. The activation barrier found for this process is 15 kcal/mol. In the transition state (TS) model, the sugar donor is partially cleaved from pyrophosphate, while nucleophilic oxygen O4(a) remains protonated with a low barrier hydrogen bond to the catalytic base D318. The structure of TS is characterized by the O4(a)-C1 and C1-O1 distances of 2.703 and 2.092 angstrom, respectively. When the obtained reaction sequence was used, the nature of the captured intermediate resembling the transition state structure (PDB/2FYD) was elucidated. This modeling QM/MM study has provided detailed insight into the mechanism of the Gal transfer catalyzed by beta 4Gal-T1 and has supplied further evidence for a concerted S(N)2-type displacement mechanism employed by inverting glycosyltransferases.

• 出版日期2009-8-13