HANDLING OF THIYL METALLOENZYME CENTERS FOR 95 & 140 GHZ EPR STUDIES

核糖核苷三磷酸还原酶(RTPR) 莱氏乳杆菌需要腺苷钴胺(ADOCbl)作为 辅因子催化核苷酸转化为 脱氧核苷酸。RTPR之前已经被证明催化了 ADOCbl的碳-钴键的均溶裂解，以及 由此产生的顺磁性物种的特征是 冷冻猝灭X波段和Q波段光谱(Orme-Johnson，W.Fl； 题名/责任者：The First of China/J.化学。1974、249、2338-2343。 李希特，S.；Gerfen，G.J.；Stubbe，J.Science 1996,271,477-481)。 模拟表明，这是一个双自旋位系统，其中钴离子和 硫基自由基(Gerfen，G.J.；Priht，S.；Willems，J.P.；Hoffman，B.R.； Stubbe，J.JACS 1996，18：2338-2343)，但较高的EPR频率是 需要就此和其他依赖B12的疾病得出明确的结论 酶系统。样品的准备、搬运和装载是 挑战95的小样品管(直径小于1 mm) IERC和麻省理工学院的EPR光谱仪分别为140 GHz和140 GHz。 这个样品处理问题正在得到解决。

The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii requires adenosylcobalamin (AdoCbl) as a cofactor to catalyze the conversion of nucleotides to deoxynucleotides. RTPR has previously been shown to catalyze the homolytic cleavage of the carbon-cobalt bond of AdoCbl, and the resulting paramagnetic species has been characterized by rapid freeze-quench X-band and Q-band spectroscopy (Orme-Johnson, W. Fl.; Beinert, H.; Blakeley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, G.J.; Stubbe, J. Science 1996, 271, 477-481). Simulations indicate a two-spin-site system with both cobalt ion and thiyl radical (Gerfen, G.J.; Licht, S.; Willems, J.P.; Hoffman, B.R.; Stubbe, J. JACS 1996, 18:2338-2343), but higher EPR frequencies are needed for definitive conclusions on this and other B12-dependent enzyme systems. Sample preparation, handling, and loading is challenging for the small sample tubes (less than 1 mm ID) of the 95 GHz and 140 GHz EPR spectromete rs at IERC and MIT, respectively. This sample handling problem is being addressed.