ENZYME REACTION INTERMEDIATES--A NEW APPROACH

酶作用的结构和动力学基础，主要是 蛋白水解酶，将通过(I)冷冻动力学方法进行研究 稳定真酶反应中间体；(Ii)电子核双 共振光谱学用于确定化合物的结构和构象 底物在溶液中游离并结合在酶的活性部位 催化活性反应中间体；(Iii)分子图形 以及用于指定酶-底物的立体化学关系的建模 基于光谱确定的数据的相互作用；(Iv)解释-- 根据立体电子学原理提取结构数据；以及 评估蛋白质残基的动态运动与 基于计算机的分子动力学模拟的酶催化功能。 这些方法是为了确定酶活性部位的局部结构。 动力学活性反应中间体中的残留物和底物， 评估蛋白质动态运动在扭转改变中的影响 底物结构以允许在活性部位进行紧密的、特定的结合， 并评估酶作用于一个分子的结构和电子基础 详细的分子水平。要应用的方法将产生数据以 催化确定其三维结构和构象 以一种不能以一种方式 目前用其他物理方法实现的。

The structural and dynamical basis of enzyme action, primarily of proteolytic enzymes, will be investigated by (i) cryokinetic methods to stabilize true enzyme reaction intermediates; (ii) electron nuclear double resonance spectroscopy to determine the structure and conformation of substrates free in solution and bound in enzymic active sites of catalytically competent reaction intermediates; (iii) molecular graphics and modeling to assign stereochemical relationships of enzyme-substrate interactions based on spectroscopically determined data; (iv) interpreta- tion of structural data according to stereoelectronic principles; and (v) assessment of the relationships of dynamical motion of protein residues to enzyme catalytic function by computer based molecular dynamics simulations. The methods are designed to determine local structure of enzyme active site residues and of substrates in kinetically competent reaction intermediates, to assess the influence of protein dynamical motion in torsionally altering substrate structure to allow tight, specific binding in the active site, and to assess the structural and electronic basis of enzyme action on a detailed molecular level. The methods to be applied will yield data to assign three-dimensional structure and conformation of catalytically competent reaction intermediates of enzymes in a manner that cannot be achieved at present with other physical methods.