Kinetic Properties of Allosteric Adenosine Monophosphate Nucleosidase from Azotobacter vinelandii

Abstract

Abstract Highly purified, stable preparations of adenosine monophosphate nucleosidase (EC 3.2.2.4) have been studied by initial velocity kinetics in order to determine the kinetic parameters of the enzyme and the relationships between the substrate and modifier sites. The results indicate that AMP nucleosidase contains interdependent modifier sites which can be occupied by MgATP2-, ATP4-, or MgPPi to give catalytically competent enzyme-activator complexes. MgATP2- is the most effective activator with ATP4- and MgPPi being less effective. The presence of saturating concentrations of activator increased the initial velocity from 100- to 400-fold over that in the absence of activator. The Hill coefficients for all three activator species are between 2.5 and 3.5 and are not affected by the concentration of AMP, the substrate of the reaction. The substrate sites exhibit interdependent kinetic behavior only at low activator concentrations and give linear double reciprocal plots of initial velocity as a function of AMP concentration at higher concentrations of activator. At high concentrations of the substrate, AMP, apparent substrate inhibition occurs. Kinetic results are consistent with this inhibition occurring by AMP combination at the modifier sites, thereby displacing the activator. Such inhibition occurs with each of the activators tested. Kinetic studies of the inhibition by AMP showed that the enzyme complex with AMP saturating at both the substrate and modifier sites retains a basal level of activity. This finding was substantiated by the demonstration of equivalent basal activity in the absence of activators. Kinetic studies of the activator-free AMP hydrolysis were consistent with the substrate sites maintaining a low rate of hydrolytic activity when the modifier sites are unoccupied. Saturation of the modifier sites with AMP leads to an increase in the rate of hydrolysis at the substrate sites. The enzyme is strongly inhibited by low concentrations of inorganic phosphate and by higher concentrations of sulfate. This inhibition occurs by competition with respect to the modifier site. From the kinetic parameters obtained, it is suggested that AMP nucleosidase is regulated in vivo by the intracellular ratios of MgATP2- and inorganic phosphate.