ELECTRONIC NATURE OF THE TRANSITION-STATE FOR NUCLEOSIDE HYDROLASE - A BLUEPRINT FOR INHIBITOR DESIGN

A new approach to understanding transition-state structure is presente d which involves the sequential application of experimental and comput ational methods. A family of experimentally determined kinetic isotope effects is fit simultaneously in a vibrational analysis to provide a geometric model of the transition state. The electrostatic potential s urface of the geometric model is defined by molecular orbital calculat ions to detail the electronic nature of the transition state. The meth od provides both geometric and charge information for the enzyme-stabi lized transition state. Electrostatic potential surface calculations w ere applied to the N-glycohydrolase reaction catalyzed by nucleoside h ydrolase from the trypanosome Crithidia fasciculata. A geometric model of the transition-state structure for the enzymatic hydrolysis of ino sine by nucleoside hydrolase has been established by the analysis of a family of kinetic isotope effects [Horenstein, B.A., Parkin, D.W., Es tupinan, B., & Schramm, V.L. (1991) Biochemistry 30, 10788]. The trans ition state has substantial oxycarbonium ion character, but the result s of electrostatic potential calculations indicate that the transition -state charge is distributed over the ribosyl ring rather than existin g as a localized C+-O <-) C=O+ resonance pair. The electrostatic poten tial surfaces of the substrate and enzyme-bound products differ consid erably from that of the transition state. At the transition state both hypoxanthine and ribose demonstrate regions of positive charge. The p ositive charge on the ribosyl oxycarbonium ion is moderated by associa tion with an enzyme-directed water nucleophile. The enzyme-bound produ cts contain adjacent areas of negative charge. The electrostatic poten tial surfaces provide novel insights into transition-state structure a nd the forces causing release of products. The reaction coordinate for nucleoside hydrolase can now be defined in terms of the molecular ele ctrostatic potential surface of inosine as it traverses the reaction c oordinate. Ribonolactone and 1,4-dideoxy-1,4-iminoribitol contain seve ral geometric features of the transition state, respectively, and are superior inhibitors compared to substrate, substrate analogues, or pro ducts.