Trypanosomal Nucleoside Hydrolase. Resonance Raman Spectroscopy of a Transition-State Inhibitor Complex
- 1 January 1996
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 35 (19) , 6037-6047
- https://doi.org/10.1021/bi9526544
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 C1' of the ribosyl [Horenstein, B. A., Parkin, D. W., Estupinan, B., & 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., & 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 C1' 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 C1' 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.Keywords
This publication has 10 references indexed in Scilit:
- Nonresonance Raman Difference Spectroscopy: A General Probe of Protein Structure, Ligand Binding, Enzymatic Catalysis, and the Structures of Other BiomacromoleculesAnnual Review of Biophysics, 1994
- Elucidation of the solution structure of the Escherichia coli aspartate aminotransferase-.alpha.-methyl-L-aspartate complex by isotope-edited Raman difference spectroscopyJournal of the American Chemical Society, 1993
- Raman spectroscopic studies of the effects of substrate binding on coenzymes bound to lactate dehydrogenaseJournal of the American Chemical Society, 1992
- Molecular recognition of amiloride analogs: a molecular electrostatic potential analysis. 1. Pyrazine ring modificationsJournal of Medicinal Chemistry, 1992
- Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism.Journal of Biological Chemistry, 1991
- Electrostatic potential maps at the quantum chemistry level of the active sites of the serine peptidases, α-chymotrypsin and subtilisinJournal of Theoretical Biology, 1990
- Use of the electrostatic potential at the molecular surface to interpret and predict nucleophilic processesThe Journal of Physical Chemistry, 1990
- Nucleoside hydrolases from Trypanosoma cruzi.Journal of Biological Chemistry, 1984
- Combination of theoretical ab initio and experimental information to obtain reliable harmonic force constants. Scaled quantum mechanical (QM) force fields for glyoxal, acrolein, butadiene, formaldehyde, and ethyleneJournal of the American Chemical Society, 1983
- RESONANCE RAMAN STUDIES OF VISUAL PIGMENTSAnnual Review of Biophysics and Bioengineering, 1977