Transition State Analysis of Enzymatic Reactions

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