Amidrazone Analogues of d-Ribofuranose as Transition-State Inhibitors of Nucleoside Hydrolase

Markus Boutellier, Bruce Ganem, Benjamin A. Horenstein, Alexi Semenyaka, Vern L. Schramm

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Abstract

The transition state of inosine during hydrolysis by nucleoside hydrolase has been characterized by kinetic isotope effects, bond-energy/bond-order vibrational analysis, and molecular electrostatic potential surface calculations [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788–10795; Horenstein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089–7097], The heterocyclic base is protonated and the anomeric carbon of the ribofuranosyl ring is flattened to form a transition-state with extensive oxocarbenium ion character. With their delocalized charge and flattened structures, amidrazone analogues of d-ribofuranose provide both geometric and electronic mimics of the ribosyl group at the transition-state of nucleoside hydrolase. A family of riboamidrazones was synthesized with H, phenyl, and p-nitrophenyl N-substituents. The analogues were competitive inhibitors with respect to inosine and gave Ki values of 10−5,2×10−7, and 1 × 10−8 M, respectively. (p-Nitrophenyl) riboamidrazone exhibited slow-onset, tight-binding inhibition, with an overall dissociation constant of 2 × 10−9 M. The binding is reversible with an off-rate of 3 × 10−3 s−1. Tight binding can be attributed to the close spatial match between the molecular geometry of (p-nitrophenyl)riboamidrazone and the transition-state stabilized by nucleoside hydrolase. The favorable binding interactions of the (p-nitrophenyl)riboamidrazone include oxocarbenium ion mimicry, isosteric ribosyl hydroxyls, and hydrophobic and H-bonding interactions at the nitrophenyl group. Analysis of the conformational space available to the (p-nitrophenyl)riboamidrazone indicates that the geometry that approximates the enzyme-stabilized transition state is 7–14 kcal/mol unfavorable relative to the global conformational minimum for free inhibitor. The apparent overall Kd of 2 nM represents only a fraction of the intrinsic energy available for transition-state interactions with nucleoside hydrolase. When corrected for the energy of distortion required to achieve the transition-state conformation, (p-nitrophenyl)riboamidrazone binds with an affinity near that expected for an ideal transition-state analogue.

Original languageEnglish (US)
Pages (from-to)3994-4000
Number of pages7
JournalBiochemistry
Volume33
Issue number13
DOIs
StatePublished - Apr 1 1994

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ASJC Scopus subject areas

  • Biochemistry

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