Investigation of Enzyme Catalysis: Mechanism at the Levels of Subunit Function and the Chemical Transformation

本研究的目的是：（1）了解 代谢物转运调控中的变构相互作用 在代谢途径中的顺序酶之间，（2） 了解酶的构象变化、构象变化与蛋白质构象的关系， 底物特异性和催化作用，以及（3）探索 直接代谢物转移在含氮系统中意义 多于一种类型的催化位点。 色氨酸合酶系统 来自大肠coli和革兰氏阳性菌S.鼠伤寒（双酶复合物）和酵母，以及 蓝绿藻（多酶）将使用快速 混合动力学技术结合吸光度和 荧光分光光度法和高分辨核 磁共振。 %%% 细胞代谢的效率需要 催化一系列化学变化的多酶复合物 对代谢物的影响。 效率源自不必 从外部环境中重新捕获中间体被怀疑是 这是这种高度组织化处理的主要原因。 的 提出的研究探索了一个理想的模型系统 底物的酶催化处理。 的情况下 色氨酸合酶，底物是通过一个“隧道”之间 酶活性位点。 计划中的研究将探讨 两个活性位点相互作用以优化 催化和代谢物在它们之间的转移。 大量 通过这项工作，我们将有可能了解到 复杂的蛋白质机器的工作原理以及为什么它被设计成这样。

The goals of this research are (1) to understand the roles of allosteric interactions in the regulation of metabolite transfer between sequential enzymes in a metabolic pathway, (2) to understand the relationship between enzyme conformation change, substrate specificity, and catalysis, and (3) to explore the significance of direct metabolite transfer in systems which contain more than one type of catalytic site. Tryptophan synthase systems from E. coli and S. typhimurium (bienzyme complexes) and yeast and blue-green algae (multienzymes) will be studied utilizing rapid mixing kinetic techniques in combination with absorbance and fluorescence spectrophotometry, and high resolution nuclear magnetic resonance. %%% The efficiency of cellular metabolism requires the existence of multienzyme complexes which catalyze a sequence of chemical changes on metabolites. The efficiency derived from not having to recapture intermediates from the exterior milieu is suspected to be the primary reason for such highly organized processing. The proposed research explores an ideal model system for such linked enzyme-catalyzed processing of substrates. In the case of tryptophan synthase, substrate is passed through a "tunnel" between the enzyme active sites. The studies planned will examine how the two active sites interact with each other in order to optimize catalysis and transfer of metabolites between them. A great deal will potentially be learned through this work about the way this complex protein machinery works and why it is designed as it is.