Bovigny Christophe, Degiacomi Matteo Thomas, Lemmin Thomas, Dal Peraro Matteo, Stenta Marco
École Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne, Switzerland.
J Phys Chem B. 2014 Jul 10;118(27):7457-7466. doi: 10.1021/jp411786z. Epub 2014 Jun 10.
The stereospecific oxidative decomposition of urate into allantoin is the core of purine catabolism in many organisms. The spontaneous decomposition of upstream intermediates and the nonenzymatic racemization of allantoin lead to an accumulation of (R)-allantoin, because the enzymes converting allantoin into allantoate are specific for the (S) isomer. The enzyme allantoin racemase catalyzes the reversible conversion between the two allantoin enantiomers, thus ensuring the overall efficiency of the catabolic pathway and preventing allantoin accumulation. On the basis of recent crystallographic and biochemical evidence, allantoin racemase has been assigned to the family of cofactor-independent racemases, together with other amino acid racemases. A detailed computational investigation of allantoin racemase has been carried out to complement the available experimental data and to provide atomistic insight into the enzymatic action. Allantoin, the natural substrate of the enzyme, has been investigated at the quantum mechanical level, in order to rationalize its conformational and tautomeric equilibria, playing a key role in protein-ligand recognition and in the following catalytic steps. The reaction mechanism of the enzyme has been elucidated through quantum mechanics/molecular mechanics (QM/MM) calculations. The potential energy surface investigation, carried out at the QM/MM level, revealed a stepwise reaction mechanism. A pair of cysteine residues promotes the stereoinversion of a carbon atom of the ligand without the assistance of cofactors. Electrostatic fingerprint calculations are used to discuss the role of the active site residues in lowering the pK of the substrate. The planar unprotonated intermediate is compared with the enolic allantoin tautomer observed in the active site of the crystallized enzyme. Finally, the enzymatic catalysis featured by allantoin racemase (AllR) is compared with that of other enzymes belonging to the same family.
尿酸盐立体特异性氧化分解为尿囊素是许多生物体嘌呤分解代谢的核心。上游中间体的自发分解以及尿囊素的非酶促外消旋化导致（R）-尿囊素的积累，因为将尿囊素转化为尿囊酸的酶对（S）异构体具有特异性。尿囊素消旋酶催化两种尿囊素对映体之间的可逆转化，从而确保分解代谢途径的整体效率并防止尿囊素积累。基于最近的晶体学和生化证据，尿囊素消旋酶与其他氨基酸消旋酶一起被归入不依赖辅因子的消旋酶家族。已对尿囊素消旋酶进行了详细的计算研究，以补充现有的实验数据并提供对酶促作用的原子水平见解。该酶的天然底物尿囊素已在量子力学水平上进行了研究，以阐明其构象和互变异构平衡，这在蛋白质-配体识别及后续催化步骤中起关键作用。通过量子力学/分子力学（QM/MM）计算阐明了该酶的反应机制。在QM/MM水平上进行的势能面研究揭示了一种逐步反应机制。一对半胱氨酸残基在没有辅因子协助的情况下促进配体碳原子的立体反转。静电指纹计算用于讨论活性位点残基在降低底物pK值方面的作用。将平面未质子化中间体与在结晶酶活性位点观察到的烯醇式尿囊素互变异构体进行比较。最后，将尿囊素消旋酶（AllR）的酶促催化作用与同一家族的其他酶进行比较。
