Adenylate degradation in Escherichia coli. The role of AMP nucleosidase and properties of the purified enzyme

Extracts of E. coli K12 degrade AMP to hypoxanthine, adenine, adenosine, and inosine. Degradation experiments with mutants which lack purine nucleoside phosphorylase or both purine nucleoside phosphorylase and adenosine deaminase demonstrate that hypoxanthine formation is dependent on purine nucleoside phosphorylase. These findings are consistent with an absence of adenine deaminase activity in E. coli. Adenine is formed from AMP in extracts of the E. coli mutants as well as the wild type cells. This activity is due to AMP nucleosidase. Purified, homogeneous AMP nucleosidase gives a subunit M(r)= 52,000 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 280,000 by comparative gel filtration. Kinetic studies with this enzyme give cooperative initial rate curves with AMP as substrate, with MgATP^2- as an activator, and with P(i) as an inhibitor. Phosphate inhibition is competitive with MgATP^2- (K(i), = 0.2 mM) and reverses the activation by MgATP^2-. In the absence of MgATP^2-, the apparent S(0.5) for AMP is 15 mM and decreases to 90 μM at saturating MgATP^2-. The maximum rate of AMP hydrolysis is not affected by MgATP^2-. Kinetics of MgATP^2- activation give a constant for half-maximum activation varying from 120 μM in the presence of low AMP to approximately 2 μM when AMP is present at near saturation. Formycin 5'-PO₄ is a powerful competitive inhibitor with respect to AMP, giving a K(is) of 72 nM and a K(m)/K(is) ratio of 1,200. Adenylate degradation experiments indicate that AMP nucleosidase is the major enzyme of AMP catabolism in E. coli. The kinetic properties of the purified enzyme indicate that regulation occurs by the intracellular MgATP^2-/P(i) ratio and the concentration of AMP.