TRANSITION STATE ANALYSIS OF ENZYMATIC REACTIONS

Yeshiva University, New York NY

Investigators

Linked publications & trials

Abstract

DESCRIPTION: The goals of this research project are to characterize the transition states of biologically significant enzymes of N-ribosyl bond scission. The transition states are used as atomic templates to develop the theory and practice of transition state inhibitor design. Transition state information and transition state analogies are used to investigate the nature of enzyme-bound transition states. The enzymes of interest include the protozoan nucleoside N-ribohydrolase isozymes and purine nucleoside phosphorylase. Protozoan parasites are purine auxotrophs and use three isozyme of N-ribohydrolases for purine salvage. The enzymes are not found in mammals. Purine nucleoside phosphorylase is widely distributed and is essential for normal T-cell function. Transition state structures for these enzymes have bee determined by kinetic isotope effect measurements using normal mode semiemperical and ab initio structural analysis. The first generation of transition state inhibitors have been synthesized and will be characterized. Experimental approaches will include: 1) laser and infrared spectroscopic investigation of the bound calcium-water center in the nucleoside hydrolases; NMR and Raman spectra of bound transition state inhibitors using isotope-edite difference analysis; and free-electron induced infrared lasers to study the actual enzymatic transition state; 2) x-ray crystallography of complexes with substrate and transition state analogues to define changes in protein structur in empty enzyme, Michaelis and transition state complexes; 3) development of theory for predicting binding energies of transition state inhibitors using trained pattern recognition; synthesis and testing of likely transition state inhibitors. The most powerful inhibitors will be tested for function in mice. The results of these studies are expected to give novel information about the nature of enzymatic transition states, provide new theory for the design of transition state inhibitors, and to provide powerful transition state inhibitors for several clinically relevant disorders.