We have performed quantum mechanics (QM), molecular mechanics (MM) and hybrid QM/MM calculations to study the stereospecific proton exchange of glutathiohydroxyacetone (HOC-SG) by glyoxalase I (GlxI). We did the QM/MM calculations with a large QM system (246 atoms) to investigate the proton-exchange mechanism. Moreover, single-point big-QM energies with 1303 atoms in the big QM system and 22,412 atoms in the MM system were used to compare the energy difference of the stationary structures. GlxI catalyzes the exchange of the pro-S, but not the pro-R hydroxymethyl proton of HOC-SG with a deuterium from the D2O solvent. Classical molecular dynamics simulations with different protonation states of Glu99, Glu172 and HOC-SG led to the determination of most stable species (Glu-172 is protonated and the alcoholic oxygen of HOC-SG is deprotonated). The QM/MM results showed that before binding of HOC-SG, both active-site glutamates are charged, whereas HOC-SG is protonated. When HOC-SG binds, its alcoholic proton (HO) can point toward either Glu-99 or Glu-172. However, if the substrate binds so that HO is directed toward Glu-99, it is not transferred, whereas if it is directed toward Glu-172, the latter abstracts HO. The results showed that transferring HO to the glutamates from the reactant states is the key step to make the proton exchange reaction possible. Our calculations show that order of basicity of the glutamates and HOC-SG inside the enzyme is: Glu-172 > HOC-SG > Glu-99. The calculations allow us to propose a reaction mechanism for the stereospecific proton exchange of HOC-SG by GlxI with an overall barrier of 14.1 kcal/mol.
