DFT studies on key intermediates in thiamin catalysis

The diphosphate ester (ThDP) of thiamin (vitamin B,) is an important cofactor of enzymes within the carbohydrate metabolism. From experiments of site-specific variants and nuclear magnetic resonance (NMR) studies, it is known that the protonation of the N1' atom is a significant step in the coenzyme activation by the enzymatic environment. Therefore, we have performed density functional theory (DFT) calculations on the B3LYP/6-31G* level of N1'H and N1'CH3 thiamin as model systems to study the protonation and methylation effect on the structure and the electronic properties of the 4'-amino group. The relaxed rotational barriers related to the C4'-4'N bond are correlated with findings of H-1 NMR studies and proton/deuterium exchange experiments. Moreover, the effect of N1' protonation was studied in more detail on the hydroxyethyl-thiamin carbanion (HETh-), a key intermediate during catalysis of some ThDP-dependent enzymes. The relaxed rotational barriers related to the C2-C2alpha bond and the reaction coordinates of the proton transfer 4'N-H --> C2alpha of HETh- and N1'H-HETh- show that they are significantly determined by the protonation at N1' of HETh-. The influence of the apoenzyme environment on the active coenzyme conformation is modeled in a very simple way. The characteristic torsion angles Phi(T) and Phi(P) are considered to be restricted in terms of their values in the corresponding enzyme as well as free optimization parameters. Frequency calculations were performed to characterize the minima and transition state structures, respectively. The applicability of the DFT method was checked by comparing calculations on the MP2-HF-SCF/6-31G* level. (C) 2004 Wiley Periodicals, Inc.