Isotope Exchange Probes of Regulatory Enzymes

大肠杆菌的天冬氨酸转甲氨基酰基酶（ATCase）是一种经典的变构酶，催化了嘧啶生物合成的第一步。抑制剂（CTP & UTP）和活化剂（ATP）结合在r链位点，传递构象信号到60A以外的c链催化位点。在过去的十年中，T态和r态酶的高分辨率x射线结构极大地提高了我们利用这一复杂系统解决复杂结构-功能问题的能力。潜在重要残基的位点特异性突变已经产生了50多种不同的物种。通过仔细选择特定类型的突变体，可以解决特定的机制问题，并测试ATCase调节的当前假设。ATCase的动力学行为将主要通过化学平衡下的同位素交换动力学（EIEK）进行研究，这被证明是定义由结合修饰剂或位点特异性突变引起的特定速率常数变化的强大而有洞察力的方法。最近已经开发了两种新方法，可以快速，深入地对各种ATCase突变体进行动力学筛选：(a)一种更快，更用户友好的ISOBI方法，(b)一种更快，更容易的交换反应，{14C}CP-CAsp，用于代替费力的{32P}- cp - P方法。为了用这个复杂的系统交叉检查和验证EIEK数据，将使用其他动力学方法的结果，包括停止流动动力学（与K.A. Johnson合作），以确定特定的速率常数，以及动力学同位素效应（与M.H. O'Leary合作）。酶是调节所有生物分子的生物合成和降解的生物催化剂。对关键调节酶的结构和功能的详细了解对于了解疾病状态和设计合理的治疗方法至关重要。胁迫这种酶的一种方法是在选定的氨基酸残基上引入特定位点的突变。一旦这样做了，就必须用动力学方法对突变酶进行深入表征。本实验室专门研究的动力学方法是化学平衡中的同位素交换。由于它能够同时观察两个方向上的快速和缓慢步骤，这种方法非常适合于精确定义哪些动力学步骤被突变改变。数据拟合是由新的、用户友好的计算机模拟程序完成的。最终，动态变化必须与x射线晶体研究和蛋白质构象变化的分子模型得出的结构数据相关联。

Wedler 9319035 Aspartate transcarbamylase (ATCase) of E. coli, a classical allosteric enzyme, catalyzes the first step of pyrimidine biosynthesis. Inhibitors (CTP & UTP) and activator (ATP) bind at r-chain sites and transmit conformational signals to separate c-chain catalytic sites over 60A away. In the past decade, high resolution x-ray structures of T- and R-state enzyme have vastly enhanced our ability to address complex structure-function questions with this complex system. Site-specific mutation of potentially important residues has yielded over 50 different species. With careful selection of mutants for certain types of intriguing changes in kinetic properties, one can address specific mechanistic questions and test current hypotheses for ATCase regulation. Dynamic behavior of ATCase will be investigated primarily by isotopic exchange kinetics at chemical equilibrium (EIEK), proven to be powerful and insightful for defining changes in specific rate constants caused by bound modifiers or site- specific mutations. Two new methods have been developed recently that permit rapid, in-depth kinetic screening of a variety of ATCase mutants: (a) a faster, more user-friendly version of the ISOBI methods, and (b) a faster, more facile exchange reaction, {14C}CP-CAsp, used in place of the laborious {32P}-CP - P, method. To cross-check and validate EIEK data with this complex system, results from other kinetic methods will be used, including stopped- flow kinetics (with K.A. Johnson) to determine specific rate constants, and kinetic isotope effects (in collaboration with M.H. O'Leary). %%% Enzymes are biological catalysts that regulate the biosynthesis and degradation of all biomolecules. A detailed knowledge of the structure-function of key regulatory enzymes is essential for understanding disease states and in designing rational therapies. One means of stressing such enzymes is the introduction of site specific mutations at selected amino acid residues. O nce this is done, the mutant enzymes must be characterized in-depth by kinetic methods. The kinetic approach in which this laboratory specializes is isotope exchange at chemical equilibrium. Due to its ability to observe the fast and slow steps in both directions simultaneously, this method is uniquely suited for defining exactly which kinetic steps are altered by a mutation. Data fitting is accomplished by new, user-friendly computer simulation programs. Ultimately, the dynamic changes must be correlated with structural data derived from x-ray crystal studies and molecular modeling of protein conformational changes.