Transition-State Structures for N-Glycoside Hydrolysis of AMP by Acid and by AMP Nucleosidase in the Presence and Absence of Allosteric Activator

Frank Mentch, David W. Parkin, Vern L. Schramm

Research output: Contribution to journalArticle

102 Scopus citations

Abstract

The mechanism of acid and enzymatic hydrolysis of the N-glycosidic bond of AMP has been investigated by fitting experimentally observed kinetic isotope effects [Parkin, D.W., & Schramm, V.L. (1987) Biochemistry (preceding paper in this issue)] to calculated kinetic isotope effects for proposed transition-state structures. The sensitivity of the transition-state calculations was tested by varying the transition-state structure and comparing changes in the calculated kinetic isotope effects with the experimental values of the isotope effect measurements. The kinetic isotope effects for the acid-catalyzed hydrolysis of AMP are best explained by a transition state with considerable oxycarbonium character in the ribose ring, significant bonding remaining to the departing adenine ring, participation of a water nucleophile, and protonation of the adenine ring. A transition-state structure without preassociation of the water nucleophile cannot be eliminated by the data. Enzymatic hydrolysis of the N-glycosidic bond of AMP by AMP nucleosidase from Azotobacter vinelandii was analyzed in the absence and presence of MgATP, the allosteric activator that increases Kmaxapproximately 200-fold. The transition states for enzyme-catalyzed hydrolysis that best explain the kinetic isotope effects involve early SN1 transition states with significant bond order in the glycosidic bond and protonation of the adenine base. The enzyme enforces participation of an enzyme-bound water molecule, which has weak bonding to Cl’ in the transition state. Activation of AMP nucleosidase by MgATP causes the bond order of the glycosidic bond in the transition state to increase significantly. Hyperconjugation in the ribosyl group is altered by enzymatic stabilization of the oxycarbonium ion. This change is consistent with the interaction of an amino acid on the enzyme. Together, these changes stabilize a carboxonium-like transition-state complex that occurs earlier in the reaction pathway than in the absence of allosteric activator. In addition to the allosteric changes that alter transition-state structure, the presence of other inductive effects that are unobserved by kinetic isotope measurements is also likely to increase the catalytic rate.

Original languageEnglish (US)
Pages (from-to)921-930
Number of pages10
JournalBiochemistry
Volume26
Issue number3
DOIs
StatePublished - 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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