Higher Flexibility of Glu-172 Explains the Unusual Stereospecificity of Glyoxalase I

Abstract

Despite many studies during the latest two decades, the reason for the unusual stereospecificity of glyoxalase I (GlxI) is still unknown. This metalloenzyme converts both enantiomers of its natural substrate to only one enantiomer of its product. In addition, GlxI catalyzes reactions involving some substrate and product analogues with a stereospecificity similar to that of its natural substrate reaction. For example, the enzyme exchanges the pro- S, but not the pro- R, hydroxymethyl proton of glutathiohydroxyacetone (HOC-SG) with a deuterium from DO. To find some clues to the unusual stereospecificity of GlxI, we have studied the stereospecific proton exchange of the hydroxymethyl proton of HOC-SG by this enzyme. We employed density functional theory and molecular dynamics (MD) simulations to study the proton exchange mechanism and origin of the stereospecificity. The results show that a rigid cluster model with the same flexibility for the two active-site glutamate residues cannot explain the unusual stereospecificity of GlxI. However, using a cluster model with full flexibility of Glu-172 or a larger model with the entire glutamates, extending the backbone into the neighboring residues, the results showed that there is no way for HOC-SG to exchange its protons if the alcoholic proton is directed toward Glu-99. However, if the hydroxymethyl proton instead is directed toward the more flexible Glu-172, we find a catalytic reaction mechanism for the exchange of the H proton by a deuterium, in accordance with experimental findings. Thus, our results indicate that the special stereospecificity of GlxI is caused by the more flexible environment of Glu-172 in comparison to that of Glu-99. This higher flexibility of Glu-172 is also confirmed by MD simulations. We propose a reaction mechanism for the stereospecific proton exchange of the hydroxymethyl proton of HOC-SG by GlxI with an overall energy barrier of 15 kcal/mol.

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.…”

“…61,62 In our previous studies on this enzyme with a rather small QM-cluster model, we used B3LYP functional, instead of TPSS. 25,26 Both are DFT methods, but the B3LYP calculations are more expensive and time consuming. Therefore, we used TPSS for the present QM/MM calculations with large QM systems.…”

“…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.…”

“…[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.…”