br Computational details and modeling All

Computational details and modeling All calculations were performed using the density functional B3LYP [34] method, implemented in Gaussian03 program [35]. The structures of reactants, transition states, intermediates, and products were optimized using the 6-31+G(d) basis set for the H, C, N, O and S atoms and the LANL2DZ basis set [36] for the Zn ion. Accurate energies were calculated with single-point calculations on the optimized structures using the larger 6-311++G(2d,2p) basis set for all atoms. This is the same DFT method as used by HS [6], but the basis sets are slightly larger, both those used for geometries and for energies. It is much more accurate than the HF/4-31G calculations performed by RK [5]. To consider the surroundings, solvation effects were evaluated at the B3LYP/6-31+G(d)/LANL2DZ level of theory by performing single-point calculations using the CPCM solvation model [37]. The CPCM calculations used UFF atomic radii and default water solvent parameters, but the dielectric constant was set to 4. Natural bond orbital (NBO) analysis [38], [39] was used to calculate atomic charges on the optimized structures. The NBO calculations were performed at the same level of theory as the single-point energy calculations. Frequencies of the stationary states on the potential energy surfaces were calculated to obtain zero-point energies. The frequency calculations were performed at the same level of theory as the geometry optimizations. All energies discussed in this article include zero-point energies and the electrostatic part of the solvation energy. A model of the active site of human GlxI was constructed based on the HIC-SG crystal structure of the native enzyme (Protein Data Bank entry 1QIN) [4]. The model consists of the zinc atom, and its first coordination shell amino acids (Gln-33, Glu-99, Glu-172 and His-126), as well as the inhibitor. The amino Furosemide residues were truncated so that only the side chains were kept in the model. Thus, the glutamates were represented by propionate, glutamine by propanamide and histidine by methyl-imidazole. The inhibitor was modified to a model of the substrate: The para-iodophenyl group was replaced by a methyl group, the N atom next to the iodophenyl group by a carbon atom and the −SG group by a −SH group. Hydrogen atoms were added manually. To maintain the overall structure of the active site, the carbon atoms bound to the H atoms that truncated the active site amino acids were fixed at their corresponding positions from the crystal structure during the geometry optimizations. The model and the fixed atoms are shown in Fig. 1.
Results and discussion To gain some understanding of the catalytic mechanism and stereospecificity of GlxI, we have performed DFT calculations on both the S and R enantiomers of the substrate in the active-site model. We discuss the results for each of these enantiomers in separate subsections.
Conclusion We have in this study performed DFT calculations on a model of the active site of GlxI, modeling the reactions for both the S and R forms of the substrate. Following the QM-cluster approach [30], [31], [32], [33], we fixed four atoms in the model to their crystallographic positions (Fig. 1). The results show that the coordination shell of the Zn ion in the optimized geometries is more symmetric than the HIC-SG crystal structure, with Glu-172 coordinating to the Zn ion in the optimized structure, but not in the crystal structure, probably owing to differences in the protonation states and the electronic structure of the HIC-SG inhibitor, compared to the substrate. We concluded that Glu-172 is protonated in the HIC-SG crystal structure. We have compared on an equal footing two suggested mechanisms for the reaction of the S substrate, as well as several additional mechanisms. We show that the HS mechanism gives a lower barrier than the RKCH mechanism, although it is more complicated. However, the results also show that an alternative route, giving rise to the other stereoisomer of the product, has a slightly lower barrier than the HS mechanism. In all mechanisms, the two active site glutamate residues (Glu99 and Glu172) play significant roles, transferring the protons between the substrate carbon and oxygen atoms. This is in agreement with mutation studies, showing that the Glu172Gln and Glu99Gln mutants have activities that are >105 and >104 smaller than that of the wild-type enzyme, respectively [40]. The active-site Zn ion is also important for the mechanism by stabilizing the enediolate intermediate and the corresponding transition state.