Mustata Gabriela Iurcu, Soares Thereza A, Briggs James M
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA.
Biopolymers. 2003 Oct;70(2):186-200. doi: 10.1002/bip.10425.
Alanine racemase (AlaR) is a bacterial enzyme that catalyzes the interconversion of L- and D-alanine, which is an essential constituent of the peptidoglycan layer of the bacterial cell wall and requires pyridoxal 5'-phosphate (PLP) as a cofactor. The enzyme is universal to bacteria, including mycobacteria, making it an attractive target for drug design. To investigate the effects of flexibility on the binding modes of the substrate and an inhibitor and to analyze how the active site is affected by the presence of the substrate versus inhibitor, a molecular dynamics simulation on the full AlaR dimer from Bacillus stearothermophilus (pdb code: 1SFT) with a D-alanine molecule in one active site and the noncovalent inhibitor, propionate, in the second site has been carried out. Within the time scale of the simulation, we show that the active site becomes more stabilized in the presence of substrate versus inhibitor. The results of this simulation are in agreement with the proposed mechanism of alanine racemase reaction in which the substrate carboxyl group directly participates in the catalysis by acting cooperatively with Tyr 265' and Lys 39. A structural water molecule in contact with both substrate and inhibitor (i.e., in both active sites) and bridging residues in both active sites was identified. It shows a remarkably low mobility and does not exchange with bulk water. This water molecule can be taken into account for the design of specific AlaR inhibitors by either utilizing it as a bridging group or displacing it with an inhibitor atom. The results presented here provide insights into the dynamics of the alanine racemase in the presence of substrate/inhibitor, which will be used for the rational design of novel inhibitors.
丙氨酸消旋酶（AlaR）是一种细菌酶，可催化L-丙氨酸和D-丙氨酸的相互转化，D-丙氨酸是细菌细胞壁肽聚糖层的重要组成部分，并且该反应需要磷酸吡哆醛（PLP）作为辅因子。这种酶在包括分枝杆菌在内的细菌中普遍存在，这使其成为药物设计的一个有吸引力的靶点。为了研究柔性对底物和抑制剂结合模式的影响，并分析活性位点如何受到底物与抑制剂存在的影响，我们对嗜热脂肪芽孢杆菌的完整AlaR二聚体（pdb代码：1SFT）进行了分子动力学模拟，其中一个活性位点含有D-丙氨酸分子，第二个位点含有非共价抑制剂丙酸酯。在模拟的时间尺度内，我们发现与抑制剂相比，底物的存在使活性位点更加稳定。该模拟结果与丙氨酸消旋酶反应的 proposed机制一致，即底物羧基通过与Tyr 265'和Lys 39协同作用直接参与催化。我们确定了一个与底物和抑制剂都接触（即在两个活性位点）的结构水分子以及两个活性位点中的桥连残基。它表现出极低的流动性，并且不与体相水交换。通过将其用作桥连基团或将其用抑制剂原子取代，可以将这个水分子考虑用于设计特异性AlaR抑制剂。此处呈现的结果为底物/抑制剂存在下丙氨酸消旋酶的动力学提供了见解，这将用于新型抑制剂的合理设计。
