Catalytic and allosteric mechanism of AMP nucleosidase from primary,. beta. -secondary, and multiple heavy atom kinetic isotope effects
Adenosine 5'-phosphate was synthesized with specific heavy atom substitutions to permit measurement of V/K kinetic isotope effects for the N-glycohydrolase activity of the allosteric AMP nucleosidase and the acid-catalyzed solvolysis of these compounds. The effects of allosteric activation on the kinetic isotope effects together with the kinetic mechanism of AMP nucleosidase indicate that the kinetic isotope effects are fully expressed. Comparison of individual primary and secondary kinetic isotope effects with combined isotope effects and the isotope effect of the reverse reaction indicated that kinetic isotope effects in AMP nucleosidase arise from a single step in the reaction mechanism. Under these conditions, kinetic isotope effects can be used to interpret transition-state structure for AMP nucleosidase. Kinetic isotope effects, expressed as (V/K(normal isotope)/(V/K(heavy isotope)), were observed with (2'-/sup 2/H)AMP, (9-/sup 15/N)AMP, (1'-/sup 2/H)AMP, and (1'-/sup 14/C)AMP when hydrolyzed by AMP nucleosidase in the absence of MgATP. Addition of MgATP altered the (2'-/sup 2/H)AMP effect and the 1'-/sup 2/H)AMP effect and cause a smaller decrease of the /sup 14/C and /sup 15/N effects. Multiple heavy atom substitutions into AMP caused an increase in observed isotope effects for (1'-/sup 2/H,1'-/sup 14/C)AMP and for (9-/sup 15/N,1'-/sup 14/C)AMP with the enzyme in the absence of ATP. The isotope effects indicate that the transition-state complex of AMP nucleosidase is oxycarbonium-like with hindered out-of-plane motion of the C1' hydrogen. In the presence of allosteric activator the bonding to C1' of ribose 5-phosphate in the transition state is unchanged, but the out-of-plane bending of the C1' and C2' hydrogens is further restricted.
- Research Organization:
- Temple Univ. School of Medicine, Philadelphia, PA
- OSTI ID:
- 6793119
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:3; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
Similar Records
Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator
Heavy atom /sup 15/N kinetic isotope effects on N-glycoside hydrolysis of AMP
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
AMP
BACTERIA
BIOSYNTHESIS
CARBOHYDRATES
CARBON 14 COMPOUNDS
DEUTERIUM COMPOUNDS
ENZYMES
ESCHERICHIA COLI
ESTERASES
GLUCOSE
HEXOSES
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPE EFFECTS
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MICROORGANISMS
MONOSACCHARIDES
NITROGEN 15
NITROGEN ISOTOPES
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PHOSPHATASES
SACCHARIDES
STABLE ISOTOPES
SYNTHESIS
TRITIUM COMPOUNDS