Adenylate degradation in Escherichia coli. The role of AMP nucleosidase and properties of the purified enzyme.
Extracts of Escherichia 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 Mr = 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 MgATP2- as an activator, and with Pi as an inhibitor. Phosphate inhibition is competitive with McATP2- (Ki = 0.2 mM) and reverses the activation by MgATP2-. In the absence of MgATP2-, the apparent S0.5 for AMP is 15 mM and decreases to 90 microM at saturating MgATP2-. The maximum rate of AMP hydrolysis is not affected by MgATP2-. Kinetics of MgATP2- activation give a constant for half-maximum activation varying from 120 microM in the presence of low AMP to approximately 2 microM when AMP is present at near saturation. Formycin 5'-PO4 is a powerful competitive inhibitor with respect to AMP, giving a Kis of 72 nM and a Km/Kis 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 MgATP2- /Pi ratio and the concentration of AMP.
D000225:Adenine / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007042:Hypoxanthines / Q000378:metabolism; D007288:Inosine / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology
D007042:Hypoxanthines; D010710:Phosphates; D000249:Adenosine Monophosphate; D007288:Inosine; D009699:N-Glycosyl Hydrolases; C019699:AMP nucleosidase; D000225:Adenine; D000241:Adenosine