Trypanosomal nucleoside hydrolase. Resonance Raman spectroscopy of a transition-state inhibitor complex

Hua Deng, Anita W.Y. Chan, Carey K. Bagdassarian, Bernardo Estupiñán, Bruce Ganem, Robert H. Callender, Vern L. Schramm

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

The transition state for hydrolysis of the N-ribosidic bond of inosine by nucleoside hydrolase has oxocarbenium character and a protonated leaving group hypoxanthine with an sp2-hybridized Cl' of the ribosyl [Horenstein, B. A., Parkin, D. W., Estupinan, B., and Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. These features are incorporated into N-(p-nitrophenyl)-D- riboamidrazone, a transition-state analogue which binds with a dissociation constant of 2 nM [Boutellier, M., Horenstein, B. A., Semenyaka, A., Schramm, V. L., and Ganem, B. (1994) Biochemistry 33, 3994-4000]. Resonance Raman and ultraviolet-visible absorbance spectroscopy has established that the inhibitor binds as the neutral, zwitterionic species. The enzyme stabilizes a specific resonance state characterized by the quinonoid form of the p- nitrophenyl group with evidence for ion pairing at the nitro group. Incorporation of 15N into a specific position of the amidrazone reveals that the exo-ribosyl nitrogen bonded to the Cl' position carries the proton while that bonded to the p-nitrophenyl carbon is unprotonated. This tautomer carries a distributed positive charge centered at the position analogous to Cl' of the ribosyl group at the transition state. The molecular electrostatic potentials for the substrate inosine, the transition state, and the transition-state inhibitor are compared at the van der Waals surface of the molecules. The tautomer of the inhibitor bound to the enzyme bears a striking electrostatic resemblance to the transition state determined by kinetic isotope effect analysis. The spectral and resonance Raman properties of free and enzyme-bound inhibitor have permitted tautomeric assignment of these species and establish that the enzyme substantially changes the electronic distribution of the bound inhibitor toward that of the enzyme-stabilized transition state.

Original languageEnglish (US)
Pages (from-to)6037-6047
Number of pages11
JournalBiochemistry
Volume35
Issue number19
DOIs
StatePublished - May 14 1996

ASJC Scopus subject areas

  • Biochemistry

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