title: Electronic nature of the transition state for nucleoside hydrolase. A blueprint for inhibitor design.
abstract: A new approach to understanding transition-state structure is presented which involves the sequential application of experimental and computational 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 surface of the geometric model is defined by molecular orbital calculations to detail the electronic nature of the transition state. The method provides both geometric and charge information for the enzyme-stabilized transition state. Electrostatic potential surface calculations were applied to the N-glycohydrolase reaction catalyzed by nucleoside hydrolase from the trypanosome Crithidia fasciculata. A geometric model of the transition-state structure for the enzymatic hydrolysis of inosine by nucleoside hydrolase has been established by the analysis of a family of kinetic isotope effects [Horenstein, B.A., Parkin, D.W., Estupinan, B., & Schramm, V.L. (1991) Biochemistry 30, 10788]. The transition state has substantial oxycarbonium ion character, but the results of electrostatic potential calculations indicate that the transition-state charge is distributed over the ribosyl ring rather than existing as a localized C+-O<==>C = O+ resonance pair. The electrostatic potential surfaces of the substrate and enzyme-bound products differ considerably from that of the transition state. At the transition state both hypoxanthine and ribose demonstrate regions of positive charge. The positive charge on the ribosyl oxycarbonium ion is moderated by association with an enzyme-directed water nucleophile. The enzyme-bound products contain adjacent areas of negative charge. The electrostatic potential surfaces provide novel insights into transition-state structure and the forces causing release of products.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461.
authors.forename: B A
authors.initials: BA
authors.lastname: Horenstein
authors.forename: V L
authors.initials: VL
authors.lastname: Schramm
mesh_terms: D000818:Animals; D001089:Arabinose; D017076:Computer-Aided Design; D016831:Crithidia fasciculata / Q000201:enzymology; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000737:chemistry*; D050113:Imino Furanoses; D007288:Inosine / Q000737:chemistry; D007700:Kinetics; D007783:Lactones / Q000737:chemistry*; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011487:Protein Conformation; D012255:Ribitol; D012266:Ribose / Q000031:analogs & derivatives* / Q000737:chemistry; D013402:Sugar Alcohols / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D050113:Imino Furanoses; D007783:Lactones; D013402:Sugar Alcohols; C058137:1,4-dideoxy-1,4-iminoarabinitol; C057020:ribonolactone; D012255:Ribitol; D007288:Inosine; D012266:Ribose; D001089:Arabinose; D009699:N-Glycosyl Hydrolases; C096466:inosine-uridine hydrolase