AMP nucleosidase

Kinetic mechanism and thermodynamics

Walter E. DeWolf, Frances A. Emig, Vern L. Schramm

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

The kinetic mechanism of AMP nucleosidase (EC 3.2.2.4; AMP + H2O → adenine + ribose 5-phosphate) from Azotobacter vinelandii is rapid-equilibrium random by initial rate studies of the forward and reverse reactions in the presence of MgATP, the allosteric activator. Inactivation-protection studies have established the binding of adenine to AMP nucleosidase in the absence of ribose 5-phosphate. Product inhibition by adenine suggests a dead-end complex of enzyme, AMP, and adenine. Methanol does not act as a nucleophile to replace H2O in the reaction, and products do not exchange into substrate during AMP hydrolysis. Thus, the reactive complex has the properties of concerted hydrolysis by an enzyme-directed water molecule rather than by formation of a covalent intermediate with ribose 5-phosphate. The Vmax in the forward reaction (AMP hydrolysis) is 300-fold greater than that in the reverse reaction. The Keq for AMP hydrolysis has been experimentally determined to be 170 M and is in reasonable agreement with Keq values of 77 and 36 M calculated from Haldane relationships. The equilibrium for enzyme-bound substrate and products strongly favors the enzyme-product ternary complex ([enzyme-adenine ribose 5-phosphate]/[enzyme-AMP] = 480). The temperature dependence of the kinetic constants gave Arrhenius plots with a distinct break between 20 and 25°C. Above 25°C, AMP binding demonstrates a strong entropic effect consistent with increased order in the Michaelis complex. Below 20°C, binding is tighter and the entropic component is lost, indicating distinct enzyme conformations above and below 25°C. Similar effects are seen on the energy of activation, which has ΔH of 11.4 kcal/mol at 30°C and 19.1 kcal/mol at 10 °C. These thermodynamic parameters have been used to construct a reaction coordinate diagram for the enzyme.

Original languageEnglish (US)
Pages (from-to)4132-4140
Number of pages9
JournalBiochemistry
Volume25
Issue number14
StatePublished - 1986
Externally publishedYes

Fingerprint

AMP nucleosidase
Thermodynamics
Adenosine Monophosphate
Adenine
Kinetics
Enzymes
Hydrolysis
Azotobacter vinelandii
Arrhenius plots
Nucleophiles
Substrates
Methanol

ASJC Scopus subject areas

  • Biochemistry

Cite this

DeWolf, W. E., Emig, F. A., & Schramm, V. L. (1986). AMP nucleosidase: Kinetic mechanism and thermodynamics. Biochemistry, 25(14), 4132-4140.

AMP nucleosidase : Kinetic mechanism and thermodynamics. / DeWolf, Walter E.; Emig, Frances A.; Schramm, Vern L.

In: Biochemistry, Vol. 25, No. 14, 1986, p. 4132-4140.

Research output: Contribution to journalArticle

DeWolf, WE, Emig, FA & Schramm, VL 1986, 'AMP nucleosidase: Kinetic mechanism and thermodynamics', Biochemistry, vol. 25, no. 14, pp. 4132-4140.
DeWolf, Walter E. ; Emig, Frances A. ; Schramm, Vern L. / AMP nucleosidase : Kinetic mechanism and thermodynamics. In: Biochemistry. 1986 ; Vol. 25, No. 14. pp. 4132-4140.
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