Department of Chemistry , University of Kurdistan , P.O. Box 66175-416, Sanandaj , Iran.
Department of Theoretical Chemistry , Lund University , P.O. Box 124, SE-221 00 Lund , Sweden.
Inorg Chem. 2018 May 7;57(9):4944-4958. doi: 10.1021/acs.inorgchem.7b03215. Epub 2018 Apr 10.
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.
尽管在最近的二十年中进行了许多研究，但仍然不清楚糖氧还蛋白 I（GlxI）异常立体特异性的原因。这种金属酶将其天然底物的两种对映异构体都转化为其产物的一种对映异构体。此外，GlxI 还催化涉及一些底物和产物类似物的反应，其立体特异性类似于其天然底物反应。例如，该酶将谷胱甘肽羟羟丙酮（HOC-SG）的 pro-S，但不是 pro-R，羟甲基质子与 DO 中的氘交换。为了找到一些线索来解释 GlxI 的异常立体特异性，我们研究了该酶对 HOC-SG 的羟甲基质子的立体特异性质子交换。我们采用密度泛函理论和分子动力学（MD）模拟来研究质子交换机制和立体特异性的起源。结果表明，具有相同两个活性部位谷氨酸残基的刚性簇模型不能解释 GlxI 的异常立体特异性。然而，使用具有完全灵活性的 Glu-172 的簇模型或具有完整谷氨酸的更大模型，将主链扩展到相邻残基，结果表明如果醇质子指向 Glu-99，则 HOC-SG 无法交换其质子。但是，如果羟甲基质子转而指向更灵活的 Glu-172，则我们找到了通过氘交换 H 质子的催化反应机制，这与实验结果相符。因此，我们的结果表明，与 Glu-99 相比，Glu-172 更灵活的环境导致了 GlxI 的特殊立体特异性。MD 模拟也证实了 Glu-172 的这种更高灵活性。我们提出了 GlxI 对 HOC-SG 的羟甲基质子进行立体特异性质子交换的反应机制，总能量势垒为 15 kcal/mol。
