Godsey Michael H, Ort Stephan, Sabini Elisabetta, Konrad Manfred, Lavie Arnon
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, 900 South Ashland Avenue, Chicago, Illinois 60607, USA.
Biochemistry. 2006 Jan 17;45(2):452-61. doi: 10.1021/bi0518646.
Human deoxycytidine kinase (dCK) uses nucleoside triphosphates to phosphorylate several clinically important prodrugs in addition to its natural substrates. Although UTP is the preferred phosphoryl donor for this reaction, our previous studies reported dCK structures solely containing ADP in the phosphoryl donor binding site. To determine the molecular basis of the kinetically observed phosphoryl donor preference, we solved crystal structures of a dCK variant lacking a flexible insert (residues 65-79) but having similar catalytic properties as wild type, in complex with deoxycytidine (dC) and UDP, and in the presence of dC but the absence of UDP or ADP. These structures reveal major changes in the donor base binding loop (residues 240-247) between the UDP-bound and ADP-bound forms, involving significant main-chain rearrangement. This loop is disordered in the dCK-dC structure, which lacks a ligand at the phosphoryl donor site. In comparison with the ADP-bound form, in the presence of UDP this loop is shifted inward to make closer contact to the smaller uracil base. These structures illuminate the phosphoryl donor binding and preference mechanisms of dCK.
人类脱氧胞苷激酶（dCK）除了利用核苷三磷酸磷酸化其天然底物外，还能磷酸化几种临床上重要的前体药物。尽管UTP是该反应中首选的磷酰基供体，但我们之前的研究报道，dCK结构在磷酰基供体结合位点仅含有ADP。为了确定动力学观察到的磷酰基供体偏好的分子基础，我们解析了一种缺乏柔性插入片段（65 - 79位氨基酸残基）但具有与野生型相似催化特性的dCK变体的晶体结构，该变体与脱氧胞苷（dC）和UDP形成复合物，以及在有dC但没有UDP或ADP存在的情况下的晶体结构。这些结构揭示了UDP结合形式和ADP结合形式之间供体碱基结合环（240 - 247位氨基酸残基）的主要变化，涉及主链的显著重排。在dCK - dC结构中，该环无序，其磷酰基供体位点缺乏配体。与ADP结合形式相比，在UDP存在的情况下，该环向内移动，以便与较小的尿嘧啶碱基更紧密接触。这些结构阐明了dCK的磷酰基供体结合和偏好机制。
