Two-Substrate Glyoxalase I Mechanism: A Quantum Mechanics/Molecular Mechanics Study

Glyoxalase I (GlxI) is an important enzyme that catalyzes the detoxification of methylglyoxal (MG) with the help of glutathione (H-SG). It is currently undear whether MG and H-SG are substrates of GlxI or whether the enzyme processes hemithioacetal (HTA), which is nonenzymatically formed from MG and H-SG. Most previous studies have concentrated on the latter mechanism. Here, we study the two-substrate reaction mechanism of GIxI from humans (Hu Gbd) and corn (ZmGlxI), which are Zn(II)active and -inactive, respectively. Hybrid quantum mechanics/molecular mechanics calculations were used to obtain geometrical structures of the stationary points along reaction paths, and big quantum mechanical systems with more than 1000 atoms and free-energy perturbations were used to improve the quality of the calculated energies. We studied, on an equal footing, all reasonable reaction paths to the S- and R-enantiomers of HTA from MG and H-SG (the latter was considered in two different binding modes). The results indicate that the MG and H-SG reaction in both enzymes can follow the same path to reach S-HTA. However, the respective overall barriers and reaction energies are different for the two enzymes (6.1 and -9.8 kcal/mol for HuGbd and 15.7 and -2.2 kcal/mol for ZmGlxI). The first reaction step to produce SHTA is facilitated by a crystal water molecule that forms hydrogen bonds with a Glu and a Thr residue in the active site. The two enzymes also follow similar paths to R-HTA. However, the reactions reach a deprotonated and protonated R-HTA in the human and corn enzymes, respectively. The production of deprotonated R-HTA in HuGbcI is consistent with other theoretical and experimental works. However, our calculations show a different behavior for ZmGbd (both S- and R-HTA can be formed in the enzyme with the alcoholic proton on HTA). This implies that Glu-144 of corn GIxI is not basic enough to keep the alcoholic proton. In HuGbd, the two binding modes of H-SG that lead to S- and R-HTA are degenerate, but the barrier leading to R-HTA is lower than the barrier to S-HTA. On the other hand, ZmGlxI prefers the binding mode, which produces S-HTA; this observation is consistent with experiments. Based on the results, we present a modification for a previously proposed two-substrate reaction mechanism for ZmGlxI.

Automated summary

This study investigated the reaction mechanism of GlxI enzymes from humans and corn, finding that both enzymes can produce the same product from MG and H-SG, but with different reaction energies and barriers. The results suggest potential modifications to the previously proposed two-substrate reaction mechanism for corn GlxI.