TRANSITION STATE ANALYSIS OF ENZYMATIC REACTIONS

Enzymatic reactions involve the formation of one or more metastable transition states on the pathway from substrates to products. Transition states of enzymatic reactions are characterized by tightly bound complexes which can be in equilibrium with the substrate or may be committed to product formation. Recent advances in the application of heavy-atom kinetic isotope effects have permitted the modeling of transition state structures for enzyme catalyzed reactions based on experimentally determined kinetic isotope effects. The purpose of this research is to determine transition state structures for purine nucleoside phosphorylase and inosine hydrolase, enzymes which catalyze phosphorolysis or hydrolysis of the H-glycosidic bond of purine nucleosides. Deficiency of purine nucleoside phosphorylase results in an immunodeficiency disorder in humans. Inosine hydrolase is an enzyme of purine salvage found in tryanosomes but not in humans. Trypanosomes have no de novo purine biosynthesis and thus depend on salvage pathways. The characterization of the transition states for these enzymes should prove useful in the development of transition state analogues for these enzymes. The transition state structure for purine nucleoside phosphorylase will be established from the family of kinetic isotope effects for the arsenolysis reaction and for the reaction with poor substrates. The transition state structure will be modeled into the crystal structure recently established for purine human erythrocyte nucleosidase. The atomic motions required to convert the reactant enzyme-substrate complex into the transition state complex should be evident from the two structures. A tightly bound intermediate which is formed between purine and purine nucleoside phosphorylase in the absence of phosphate will be characterized. Inosine hydrolase will be isolated from the trypanosome Crithidia fasciculata. Following transition state analysis by heavy atom kinetic isotope effects, the enzyme will be cloned from a genomic DNA library from C. fasciculata library, sequenced and expressed in E. coli. Attempts will be made to crystallize the protein for the eventual goal of correlating the transition state structure and the catalytic site structure.