Glyoxalase I (GlxI) is an important
enzyme that catalyzes the detoxification of methylglyoxal (MG) with
the help of glutathione (H-SG). It is currently unclear 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 GlxI from humans (HuGlxI) 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 HuGlxI and 15.7 and −2.2 kcal/mol
for ZmGlxI). The first reaction step to produce S-HTA 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 HuGlxI is consistent with other
theoretical and experimental works. However, our calculations show
a different behavior for ZmGlxI (both S- and R-HTA can be formed in the enzyme
with the alcoholic proton on HTA). This implies that Glu-144 of corn
GlxI is not basic enough to keep the alcoholic proton. In HuGlxI, 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.