EPR: ADENOSYLCOBALAMIN DEPENDENT ENZYME RIBONUCLEOTIDE TRIPHOSPHATE REDUCTASE

核糖核苷酸三磷酸还原酶（RTPR）。莱希曼尼 催化腺苷钴胺素依赖性的核苷酸还原， 脱氧核苷酸 当酶快速与 腺苷钴胺素，变构效应物和还原系统， 顺磁性物质以动力学上胜任的方式产生。 虽然最初观察到20多年前， 自由基物种是以前未知的。 半胱氨酸的同位素标记 结合对该系统的模拟， 这个信号与一个硫代自由基相互作用的信号是一致的， 钴（II）丙氨酸。 然而，在这方面仍有许多问题有待了解。 这种硫基自由基-钴（II）丙氨酸对的结构。 高频 EPR和ENDOR光谱将用于深入了解 自由基对的分子和电子结构。 细化 未成对电子之间的距离和相对取向 的thiyl自由基和cob（II）alamin对将给予额外的 结构酶活性位点的额外结构细节。 一 关键问题涉及脱氧腺苷部分的ATE，因为 5 '脱氧腺苷基最初形成于Co-C键上 未观察到均裂。 脉冲EPR/ENDOR结合 在5 '脱氧腺苷上的不同位置的13 C和2 H标记将 确定其在活性部位中的位置。 此外，RTPR是 在与几种不同的核苷酸孵育后容易抑制 类似物通过未知的自由基为基础的机制。 脉冲EPR、ENDOR和 将进行高频EPR光谱分析， 这些自由基中间体，并确定RTPR的机制 抑制作用

Ribonucleotide triphosphate reductase (RTPR) from L. leichmannii catalyzes the adenosylcobalamin-dependent reduction of nucleotides to deoxynucleotides. When the enzyme is rapidly mixed with adenosylcobalamin, an allosteric effector and a reducing system, a paramagnetic species is generated in a kinetically competent fashion. Although originally observed over 20 years ago, the identity of the radical species was previously unknown. Isotopic labeling of cystein in conjunction with simulations of this system have recently shown that the signal is consistent with a thiyl radical interacting with cob(II)alamin. However, much remains to be learned concerning the structure of this thiyl radical-cob(II)alamin pair. High frequency EPR and ENDOR spectroscopies will be used to give insight into the molecular and electronic structure of the radical pair. Refinement of the distance between unpaired electrons and the relative orientation of the thiyl radical and cob(II)alamin pair will give additional structural additional structural detail to the enzyme active site. A key question concerns the ate of the deoxyadenosyl moiety, since the 5'deosyadenosyl radical proposed to initially form upon Co-C bond homolysis has not been observed. Pulsed EPR/ENDOR in conjunction with 13C and 2H labeling at various positions on the 5'deoxyadenosine will determine its placement in the active site. In addition, RTPR is readily inhibited upon incubation with several different nucleotide analogs via unknown radical based mechanisms. Pulsed EPR, ENDOR and high frequency EPR spectroscopy will be performed in order to identify these radical intermediates and to determine the mechanisms of RTPR inhibition.