EPR: ADENOSYLCOBALAMIN DEPENDENT ENZYME RIBONUCLEOTIDE TRIPHOSPHATE REDUCTASE

莱氏乳杆菌核糖核酸三磷酸还原酶(RTPR) 催化腺苷钴胺依赖的核苷酸还原为 脱氧核苷酸。当这种酶快速地与 腺苷钴胺、变构效应剂和还原系统 顺磁物种是以能动的方式产生的。 尽管最初是在20多年前观察到的，但 激进物种以前是未知的。半胱氨酸的同位素标记 结合对该系统的模拟，最近已经显示 该信号与硫基自由基相互作用一致 CoB(II)丙氨酸。然而，关于这一点，还有很多需要了解 该硫基自由基-CoB(II)丙氨酸对的结构。高频 EPR和Endor光谱分析将用于深入了解 自由基对的分子结构和电子结构。精细化 未成对电子之间的距离和相对取向 硫基自由基和CoB(II)丙胺对将给出额外的 酶活性部位的结构附加结构细节。一个 关键问题涉及脱氧腺苷部分的摄入，因为 建议在Co-C键上初步形成的5‘-脱糖腺苷自由基 未观察到均质溶解现象。脉冲EPR/Endor与 5‘脱氧腺苷上不同位置的13C和2H标记 确定其在活动站点中的位置。此外，RTPR是 在与几个不同的核苷酸孵育时容易被抑制 通过未知的基于自由基的机制进行类比。脉冲EPR、Endor和 将进行高频电子顺磁共振波谱分析以确定 这些自由基中间体，并确定RTPR的机制 抑制力。

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.