TIME RESOLVED STUDIES OF B12 ENZYMES AN COENZYMES

说明：（申请人摘要）维生素B12是人体必需的 作为腺苷钴胺素依赖的辅酶所需的营养素 甲基丙二酰辅酶A和甲基钴胺素依赖性甲硫氨酸合酶。 除了B12依赖性酶外，还有许多B12转运 参与钴酸盐的调节和传递的蛋白质。 关键 B12中的催化元素是独特的碳-钴键。 的目的 目前的研究计划是使用时间分辨光谱来阐明 蛋白质在控制和指导细胞的反应性中的作用 碳钴键 B12酶包括一类蛋白质系统 它们非常适合于应用时间分辨的 光谱技术 用可见光激发导致裂解 C-Co的能量 晶体的可用性 两种酶的B12结合结构域的结构和 过表达并产生大量的各种野生型和突变体 酶使物理方法应用于机理研究 在B12特别有希望。 建议的工作将调查光化学和物理 烷基钴烷和烷基可啉类化合物中碳-钴键的性质 在溶液中作为pH和溶剂的函数，以及作为轴向的函数， 基地 对自由辅因子的透彻理解将有助于深入了解 不稳定的C-Co键在自然界中被利用和控制的方式。 该项目还将涉及直接研究与 B12蛋白，特别是甲硫氨酸合酶和谷氨酸盐。 这些 研究将探索蛋白质的自由基笼特性。 在 此外，由蛋白质提供的轴向组氨酸配体的作用将 通过使用一系列突变来去除/替换该组氨酸来探测 并改变与组氨酸氢键结合的氨基酸残基。

DESCRIPTION: (applicant's abstract) Vitamin B12 is an essential human nutrient required as a coenzyme for adenosylcobalamin dependent methylmalonyl-CoA mutase and methylcobalamin dependent methionine synthase. In addition to the B12-dependent enzymes there are a number of B12 transport proteins involved in the regulation and delivery of cobalamins. The key catalytic element in B12 is a unique carbon-cobalt bond. The aim of the present research program is to use time-resolved spectroscopy to elucidate the role of the protein in controlling and directing the reactivity of the carbon-cobalt bond. The B12 enzymes comprise a class of protein systems which lend themselves beautifully to the application of time-resolved spectroscopic techniques. Excitation with visible light results in cleavage of the C-Co within a few picoseconds. The availability of crystal structures for the B12 binding domain of two enzymes and the ability to overexpress and produce significant amounts of various wild-type and mutant enzymes makes the application of physical methods to the study of mechanism in B12 particularly promising. The work proposed will investigate the photochemical and photophysical properties of the carbon-cobalt bond in alkylcobalamins and alkylcorrinoids in solution as a function of pH and solvent, and as a function of the axial base. A thorough understanding of the free cofactors will lend insight into the ways that the labile C-Co bond is utilized and controlled in nature. This project will also involve the direct study of alkylcobalamins bound to B12 proteins, particularly methionine synthase and glutamate mutase. These studies will explore the radical caging properties of the proteins. In addition the role of the axial histidine ligand supplied by the protein will be probed by using a series of mutations to remove/replace this histidine and to alter the amino acid residues hydrogen bonded to the histidine.