Reaction mechanism of the binuclear zinc enzyme glyoxalase II - A theoretical study.

The glyoxalase system catalyzes the conversion of toxic methylglyoxal to nontoxic d-lactic acid using glutathione (GSH) as a coenzyme. Glyoxalase II (GlxII) is a binuclear Zn enzyme that catalyzes the second step of this conversion, namely the hydrolysis of S-d-lactoylglutathione, which is the product of the Glyoxalase I (GlxI) reaction. In this paper we use density functional theory method to investigate the reaction mechanism of GlxII. A model of the active site is constructed on the basis of the X-ray crystal structure of the native enzyme. Stationary points along the reaction pathway are optimized and the potential energy surface for the reaction is calculated. The calculations give strong support to the previously proposed mechanism. It is found that the bridging hydroxide is capable of performing nucleophilic attack at the substrate carbonyl to form a tetrahedral intermediate. This step is followed by a proton transfer from the bridging oxygen to Asp58 and finally C-S bond cleavage. The roles of the two zinc ions in the reaction mechanism are analyzed. Zn2 is found to stabilize the charge of tetrahedral intermediate thereby lowering the barrier for the nucleophilic attack, while Zn1 stabilizes the charge of the thiolate product, thereby facilitating the C-S bond cleavage. Finally, the energies involved in the product release and active-site regeneration are estimated and a new possible mechanism is suggested.