Thermodynamic analysis of the oxidative deamination of polyamines by bovine serum amine oxidase.
The effect of incubation temperature on the activity of bovine serum amine oxidase was studied using natural substrates of this enzyme, namely spermine and spermidine. The activity behavior was found to be rather complex and different from that observed using benzylamine as substrate. The enzyme is fully active after 3 min incubation at temperatures up to 60 degrees C, while at higher temperatures it shows an S-shaped irreversible decrease of the activity with a T50 of about 70 degrees C. The dependence of the kinetic parameters Kcat and Km on temperature was also studied in the range 5-66 degrees C, measuring the initial rates of oxidation of spermidine. The Arrhenius plot of kcat shows a continuous bend in the thermal stability range of the enzyme. This nonlinearity is due to the dramatic change of kcat activation entropy (from +11 eu at 8 degrees C, to -31 eu at 50 degrees C) and indicates that kcat is a composite constant, involving two or more successive steps characterized by different kinetic parameters. In the case of the ratio kcat/Km, which for natural substrates of bovine serum amine oxidase was shown that its dependence on ionic strength gives information on the influence of electrostatic field on k1 (Stevanato et al. (1994) Biochem. J. 299, 317-320), where k1 is the kinetic constant of interaction between the polyamine and the enzyme, the Arrhenius plot is linear in the range 12-45 degrees C, and an activation entropy of 18.8 eu was calculated at 37 degrees C. This value is in accord with a mechanism involving the formation of a polyamine-enzyme complex which is accompanied by neutralization of charges between the polycationic substrate and negatively charged groups of the enzyme.
Stevanato
R
R
Department of Physical Chemistry, University of Venice, Italy.
Vianello
F
F
Rigo
A
A
D006631:Amine Oxidase (Copper-Containing); D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002417:Cattle; D003641:Deamination; D008667:Metalloproteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000097:blood / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism*; D011489:Protein Denaturation; D013816:Thermodynamics
D001798:Blood Proteins; D008667:Metalloproteins; D011073:Polyamines; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors