Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator

Journal Article · Tue Feb 10 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00377a037· OSTI ID:6526080

Mentch, F; Parkin, D W; Schramm, V L

The mechanism of acid and enzymatic hydrolysis of the N-glycosidic bond of AMP has been investigated by fitting experimentally observed kinetic isotope effects 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 which 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-glycoside bond of AMP by AMP nucleosidase from Azotobacter vinelandii was analyzed in the absence and presence of MgATPhe allosteric activator that increases V/sub max/ approximately 200-fold. The transition states for enzyme-catalyzed hydrolysis that best explain the kinetic isotope effects involve early S/sub N/1 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 C1' 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. 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.

• Research Organization: Temple Univ. School of Medicine, Philadelphia, PA
• OSTI ID: 6526080
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:3; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMP
AZOTOBACTER
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CHEMICAL REACTIONS
DECOMPOSITION
DEUTERIUM COMPOUNDS
ENZYMATIC HYDROLYSIS
ENZYMES
ESTERASES
HYDROGEN COMPOUNDS
HYDROLASES
HYDROLYSIS
ISOTOPE EFFECTS
KINETICS
LABELLED COMPOUNDS
LYSIS
MICROORGANISMS
NUCLEOTIDES
ORGANIC COMPOUNDS
PHOSPHATASES
REACTION KINETICS
SOLVOLYSIS
STEREOCHEMISTRY
TRITIUM COMPOUNDS