GLO1 - Mechanism

Mechanism

The methylglyoxal molecule consists of two carbonyl groups flanked by a hydrogen atom and a methyl group. In the discussion below, these two carbonyl carbons will be denoted as C₁ and C₂, respectively. In both the hemithioacetal substrate and the (R)-S-lactoylglutathione product, the glutathione moiety is bonded to the C₁ carbonyl group.

The basic mechanism of glyoxalase I is as follows. The substrate hemithioacetal is formed when a molecule of glutathione — probably in its reactive thiolate form — attacks the C₁ carbonyl of methylglyoxal or a related compound, rendering that carbon tetravalent. This reaction occurs spontaneously in the cell, without the involvement of the enzyme. This hemithioacetalis then bound by the enzyme, which shifts a hydrogen from C₁ to C₂. The C₂ carbonyl is reduced to a tetravalent alcohol form by the addition of two protons, whereas the C₁ carbonyl is restored by losing a hydrogen while retaining its bond to the glutathione moiety.

A computational study, combined with the available experimental data, suggests the following atomic-resolution mechanism for glyoxalase I. In the active site, the catalytic metal adopts an octahedral coordination geometry and, in the absence of substrate, binds two waters, two opposite glutamates, a histidine and one other sidechain, usually another histidine or glutamates. When the substrate enters the active site, the two waters are shed and the two carbonyl oxygens of the substrate are bound directly to the metal ion. The two opposing glutamates add and subtract protons from C₁ and C₂ and their respective oxygens, O₁ and O₂. The first half of the reaction transfers a proton from C₁ to O₂, whereas the second half transfers a proton from O₁ to C₂. The former reaction may be carried out by either of the opposing glutamates, depending on the initial chirality of C₁ in the hemithioacetal substrate; however, the second half is stereospecific and is carried out by only one of the opposing glutamates.