Catalytic mechanism of human glyoxalase I studied by quantum-mechanical cluster calculations

Abstract

Density functional theory has been used to study the mechanism and stereospeci7icity of the catalytic reaction of human glyoxalase I. We used the quantum mechanical cluster method to model the enzyme active site. Glyoxalase I accepts both enantiomers of the hemithioacetal between methylglyoxal and glutathione and converts them to the S-D enantiomer of lactoylglutathione. We have compared several previously suggested or alternative reaction mechanisms for both substrates on an equal footing. The results show that the coordination shell of the Zn ion in the optimized geometries is more symmetric than in some inhibitor crystal structures, which we assign to differences in the electronic structure and the protonation states. The symmetry of the active site model indicates that the enzyme can use the same reaction mechanism for the S and the R enantiomers of the substrate, but with exchanged roles of the two active-site glutamate residues. However, the calculations show some asymmetry (0-4 kcal mol -1 differences in reaction energies and activation barriers), caused by the different coordination states of the glutamate residues in the starting crystal structure. Our results indicate that the only possibility for the stereospeci7icity of GlxI is differences in the electrostatic surroundings and 7lexibility of the glutamate residues in the active site owing to their neighboring residues in the protein.

Quantum Mechanics/Molecular Mechanics Study of the Reaction Mechanism of Glyoxalase I

“…This confirms the higher basicity of Glu-172 compared to Glu-99, as was previously proposed. [23][24][25][26] The big-QM energies of the four states are compared in Table 1, showing that the S1, S2 and R2 states are almost degenerate, whereas the R1 state is ~4 kcal/mol less stable than the others. In the latter, both protons (H1 and H2) point toward Glu-99, which may destabilize it by steric effects.…”

“…In summary, the three studies indicated that the higher basicity and flexibility of Glu-172 may explain the special stereospecificity of GlxI. Despite all previous studies, [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] there is not any computationally or experimentally confirmed mechanism for the reaction of the R enantiomer of the normal substrate of GlxI. Moreover, there are two opposing mechanisms for the S substrate and they are based on either a rather primitive ab initio method (HF/4-31G) or a small model of the active site with no constraints on the residues during optimization processes.…”

“…23 Recently, we confirmed the higher basicity of this residue by quantum mechanical cluster (QM-cluster) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. [24][25][26] In addition, we showed through molecular dynamics (MD) simulations that Glu-172 has a higher flexibility than Glu-99 and this flexibility causes its displacement form the Zn ion and its higher basicity. 25 However, Hartree-Fock and density functional theory (DFT) calculations using relatively small models and symmetric glutamates could not explain the unusual specificity of GlxI.…”

“…25 However, Hartree-Fock and density functional theory (DFT) calculations using relatively small models and symmetric glutamates could not explain the unusual specificity of GlxI. [26][27][28] In the last two decades, different aspects of the catalytic mechanism of GlxI have been studied. [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] Two mechanisms were proposed for the reaction of the S substrate with GlxI in 2001.…”

“…[26][27][28] In the last two decades, different aspects of the catalytic mechanism of GlxI have been studied. [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] Two mechanisms were proposed for the reaction of the S substrate with GlxI in 2001. [27][28][29] Richter and Krauss (RK), 28 used Hartree-Fock calculations, coupled with a frozen effective fragment potential, 38,39 whereas Creighton and Hamilton (CH), 29 summarizing experimental aspects of the catalytic mechanism of GlxI up to that date, suggested independently the same three-step mechanism shown in Scheme 2.…”