Density functional theory has been used to study the mechanism and stereospecificity of the catalytic reaction of human glyoxalase I. We used the quantum mechanical cluster method to model the enzyme active site. Glyoxalase I accepts both enantiomers of the hemithioacetal between methylglyoxal and glutathione and converts them to the S-D enantiomer of lactoylglutathione. We have compared several previously suggested or alternative reaction mechanisms for both substrates on an equal footing. The results show that the coordination shell of the Zn ion in the optimized geometries is more symmetric than in some inhibitor crystal structures, which we assign to differences in the electronic structure and the protonation states of the substrate. The symmetry of the active site model indicates that the enzyme can use the same reaction mechanism for the S and the R enantiomers of the substrate, but with exchanged roles of the two active-site glutamate residues. However, the calculations show some asymmetry (0–4 kcal mol−1 differences in reaction energies and activation barriers), caused by the different coordination states of the glutamate residues in the starting crystal structure. Our results indicate that the only possibility for the stereospecificity of glyoxalase I is differences in the electrostatic surroundings and flexibility of the glutamate residues in the active site owing to their neighboring residues in the protein.
