Department of Physics, Indian Institute of Science, Bangalore 560012, India.
Department of Computational and Data Sciences, Indian Institute of Science, Bangalore 560012, India.
Int J Biol Macromol. 2019 Feb 15;123:637-647. doi: 10.1016/j.ijbiomac.2018.11.025. Epub 2018 Nov 14.
Several studies on enzyme catalysis have pointed out that the product release event could be a rate limiting step. In this study, we have compared the release event of two products, Adenosine di-phosphate (ADP) and Thymidine di-phosphate (TDP) from the active-site of human and Thermus thermophilus thymidine mono-phosphate kinase (TMPK), referred to as hTMPK and ttTMPK, respectively. TMPK catalyses the conversion of Thymidine mono-phosphate (TMP) to TDP using ATP as phosphoryl donor in the presence of Mg ion. Most of the earlier studies on this enzyme have focused on understanding substrate binding and catalysis, but the critical product release event remains elusive. Competitive binding experiments of the substrates and the products using ttTMPK apo crystals have indicated that the substrate (TMP) can replace the bound product (TDP), even in the presence of an ADP molecule. Further, the existing random accelerated molecular dynamics (RAMD) simulation program was modified to study the release of both the products simultaneously from the active site. The RAMD simulations on product-bound structures of both ttTMPK and hTMPK, revealed that while several exit patterns of the products are permissible, the sequential exit mode is the most preferred pattern for both ttTMPK and hTMPK enzymes. Additionally, the product release from the hTMPK was found to be faster and more directional as compared to ttTMPK. Structural investigation revealed that the critical changes in the residue composition in the LID-region of ttTMPK and hTMPK have an effect on the product release and can be attributed to the observed differences during product release event. Understanding of these dissimilarities is of considerable utility in designing potent inhibitors or prodrugs that can distinguish between eukaryotic and prokaryotic homologues of thymidylate kinase.
已有多项关于酶催化的研究指出，产物释放事件可能是限速步骤。在这项研究中，我们比较了人源和嗜热栖热菌胸苷单磷酸激酶（TMPK）活性位点上两种产物（二磷酸腺苷（ADP）和二磷酸胸苷（TDP））的释放事件，分别称为 hTMPK 和 ttTMPK。TMPK 在 Mg2+离子存在下，以 ATP 为磷酸供体，催化胸苷单磷酸（TMP）转化为 TDP。早期关于该酶的大多数研究都集中在理解底物结合和催化上，但关键的产物释放事件仍然难以捉摸。使用 ttTMPK 脱辅基晶体进行的底物和产物的竞争性结合实验表明，即使存在 ADP 分子，底物（TMP）也可以取代结合的产物（TDP）。此外，对现有的随机加速分子动力学（RAMD）模拟程序进行了修改，以同时研究两种产物从活性部位的释放情况。对 ttTMPK 和 hTMPK 产物结合结构的 RAMD 模拟表明，虽然几种产物的出口模式是允许的，但顺序出口模式是 ttTMPK 和 hTMPK 酶最优选的模式。此外，与 ttTMPK 相比，hTMPK 的产物释放更快且更具方向性。结构研究表明，LID 区域中残基组成的关键变化对产物释放有影响，并可归因于产物释放事件中观察到的差异。了解这些差异对于设计能够区分真核和原核胸苷酸激酶同系物的有效抑制剂或前药具有重要意义。
