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.
