The glyoxalase system catalyzes the conversion of toxic methylglyoxal to D-lactate. Glyoxalase II (GlxII) catalyzes the second step of this conversion, namely the hydrolysis of S-Dlactoylglutathione (SLG), which is the product of the Glyoxalase I (GlxI) reaction. In this study, we investigated the reaction mechanism of GlxII by the quantum mechanics/molecular mechanics (QM/MM) method. A model of the active site of GlxII was constructed based on the crystal structure of the human GlxII (1QH5 PDB ID). Its active site contains two zinc ions, an aspartic acid (Asp-58), several histidine ligands (His-54, His-56, His-59, His-110, and His-173) and a bridging Asp-134 group, a hydroxide (μ-OH), and SLG as the substrate (Figure 1). The results showed that the first step of the mechanism is the nucleophilic attack of the bridging oxygen (Oμ) on the carbonyl carbon (Cs) of the substrate (the atom names are defined in Figure 1) that concurrently with this attack, the S-Cs bond dissociation occurs. Then the mechanism proceeds with two successive proton transfers, first the transfer of H2w from a water molecule to the S atom. After that, the transfer of the Hμ to the O1 atom of the substrate produces a lactate ion. Our results indicated that the initial nucleophilic attack is the rate-limiting step which contradicts previous calculations on other dinuclear zinc enzymes.
