TIME RESOLVED STUDIES OF B12 ENZYMES AN COENZYMES

描述：(申请人摘要)维生素B12是人体必需的 腺苷钴胺依赖所需的辅酶营养 甲基丙二酰辅酶A变位酶和甲钴胺依赖的蛋氨酸合成酶。 除了依赖于B12的酶，还有许多B12的运输 与钴蛋白的调节和传递有关的蛋白质。钥匙 B12中的催化元素是一种独特的碳-钴键。该计划的目的是 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活性中的作用 碳-钴键。B12酶由一类蛋白质系统组成 它们非常适合于时间分辨的应用 光谱技术。可见光激发导致卵裂 在几皮秒内碳-钴的含量。水晶的可获得性 两种酶的B12结合域的结构及其结合能力 过量表达并产生大量的各种野生型和突变体 酶使物理方法在机理研究中的应用 在B12中特别有希望。 提出的工作将研究光化学和光物理 烷基钴胺和烷基珊瑚碱类化合物中碳-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。对自由余因式的透彻理解将有助于洞察 不稳定的C-Co键在性质上的利用和控制方式。 该项目还将涉及对烷基钴胺的直接研究 B12蛋白，特别是蛋氨酸合成酶和谷氨酸变位酶。这些 研究将探索蛋白质的自由基笼化特性。在……里面 此外，由蛋白质Will提供的轴向组氨酸配体的作用 通过使用一系列突变来移除/替换这种组氨酸来进行探测 并改变与组氨酸氢键的氨基酸残基。

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.