Enzyme structure-function studies require both dynamic and static approaches. Among the various kinetic methods, isotopic exchange at chemical equilibrium can provide a unique approach for addressing mechanistic questions. The theoretical basis and practical basis for this method was dramatically advanced recently by the PI's development of computer simulation methods for exchange data analysis. Equilibrium isotope exchange kinetics was proven to be especially useful for determining kinetic mechanism and defining which steps are altered by bound effectors or by mutation of specific amino acid residues. The enzymes proposed for study were selected for their potential to advance fundamental knowledge, the crucial regulatory role each plays in a key metabolic pathway, their complex responses to effectors, and identification of important, yet unanswered mechanistic questions that can be addressed uniquely by EIEK methods: a) Aspartate transcarbamylase regulates biosynthesis of pyrimidines needed for DNA. Substrate analogs and mutant enzymes have been selected that will allow the dissection of the mechanisms by which the effectors ATP and CTP operate and to determine how interaction of substrate with active site groups triggers the dramatic conformational change of allosteric transition. b) Phosphorylase controls energy metabolism by mobilizing stored glucose (glycogen) into the glycolytic pathway. The major challenges with this system will be determine which step (s) are altered by selected modifiers in an enzyme for which chemical catalysis, rather than substrate binding, appears to be the rate-limiting process in net turnover. Development of more advanced computer programs, to simulate complex enzyme behavior and for data fitting, is also essential to this project. Routines for linear regression analysis will also be incorporated, to optimize data fitting and to help define confidence limits for the derived rate constants.
