PROTEIN STRUCTURE AND FUNCTION

我们正在研究一种叫做甲酰四氢叶酸合成酶的酶， 酶学家感兴趣的问题的数量;机制，底物 小配体对结构的结合和控制。 概述的实验 旨在加深我们对 结构和催化活性，从而增加了我们 了解一般的酵素。 我们计划用~（13）C、~（1H）和~（31）P核磁共振弛豫技术来研究配体 约束力 这些研究将在不同的条件下进行，以确定 核与酶结合Mn（II）之间的核间距;是否 单体的结合位点存在可检测的差异， 四聚体，以及配体是否产生构象变化 约束力 紫外差光谱和荧光光谱将用于研究 ATP和ATP类似物与酶的相互作用。 各种实验 提出寻找证据的存在，酶结合甲酰基 磷酸盐作为中间体。 ATP的结合位点将通过 使用可光活化的ATP类似物的UV诱导交联，然后 蛋白水解消化。 阳离子诱导的单体再缔合反应的研究 还提出了反应。 组装机制将遵循 紫外差光谱和常规和快速动力学技术。 在一些实验中，将对梭菌和 酶，被研究的主要蛋白质，和酵母酶。 主要 原核酶和真核酶之间存在结构差异， 这使得这些比较很有趣。

We are studying the enzyme, formyltetrahydrofolate synthetase, to examine a number of problems of interest to enzymologists; mechanism, substrate binding and control of stucture by small ligands. The experiments outlined are designed to increase our understanding of the relationship between structure and catalytic activity of this enzyme and thereby add to our understanding of enzymes in general. We plan to use 13C, 1H and 31P NMR relaxation techniques to study ligand binding. The studies will be done under different conditions to determine internuclear distances between the nucleus and enzyme-bound Mn(II); whether detectable differences exist in the binding sites of the monomer and tetramer, and whether conformational changes are produced upon ligand binding. UV-difference and fluorescence spectroscopy will be used to study ATP and ATP-analog interactions with the enzyme. Various experiments are proposed to find evidence for the existence of an enzyme-bound formyl phosphate as an intermediate. The binding site for ATP will be explored by using UV-induced crosslinking of photo-activatable ATP analogs, followed by proteolytic digestion. Studies of the cation-induced monomer reassociation reaction are also proposed. The assembly mechanism will be followed by UV-difference spectroscopy and conventional and rapid kinetic techniques. In some of the experiments comparisons will be made between the clostridial enzyme, the primary protein being studied, and the yeast enzyme. Major structural differences exist between the procaryotic and eucaryotic enzyme, which makes these comparisons of interest.