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

本研究的目的是：(1)了解变构相互作用在代谢途径中序列酶之间代谢物转移调控中的作用；(2)了解酶构象改变、底物特异性和催化之间的关系。(3)探索含有多种催化位点的系统中代谢物直接转移的意义。利用快速混合动力学技术，结合吸光度和荧光分光光度法以及高分辨率核磁共振技术，研究大肠杆菌和鼠伤寒杆菌（双酶复合物）以及酵母和蓝绿藻（多酶）的色氨酸合成酶系统。细胞代谢的效率需要多酶复合物的存在，多酶复合物催化代谢产物的一系列化学变化。由于不必从外部环境中重新获取中间体而产生的效率被怀疑是这种高度组织化处理的主要原因。提出的研究探索了一个理想的模型系统，这种连接酶催化处理底物。在色氨酸合成酶的情况下，底物通过酶活性位点之间的“隧道”。计划中的研究将检查这两个活性位点如何相互作用，以优化它们之间代谢物的催化和转移。通过这项工作，我们可能会了解到很多关于这种复杂的蛋白质机制的工作方式，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.