Comparison of initial velocity and binding data for allosteric adenosine monophosphate nucleosidase.
Adensine monophosphate nucleosidase (AMP nucleosidase) from Azotobacter vinelandii is composed of six subunits with similar or identical charge and size and has a molecular weight of approximately 320,000. Binding studies with tritiated tubercidin 5' -PO4 (4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5' -monophosphate), a competitive inhibitor with respect to the substrate, AMP, indicate the presence of three independent, identical binding sites for the substrate analog. The binding of tubercidin 5' -PO4 is not affected by either Mg2+ or MgATP2-; however, in initial velocity experiments MgATP2- caused from greater than100- to 4,000-fold activation of substrate hydrolysis depending on the concentration of AMP. Binding studies with [14C]ATP are consistent with six interdependent binding sites for MgAT2-. Initial velocity and binding curves for MgATP2- are similar in shape, but reveal a disproportionate increase in initial velocity at low saturation levels of MgAT2-. Binding of MgAT2- is inhibited by increasing concentrations of P1 which acts as a competitive inhibitor of MgATP2- activation in both initial velocity and binding experiments. In the absence of MgATP2-, 32Pi binds at six or more interdependent modifier sites. The simulataneous binding of Mg[14C]ATP2- and 32Pi was studied in experiments where MgATP2- and Pi were held in constant ratio. Extrapolation to infinite concentrations of both MgATP2- and Pi indicated that 3 molecules of each were bound to the enzyme. Thus the binding of the allosteric activator and inhibitor are mutually exclusive. These results are consistent with a single modifier site per subunit at which either MgATP2- or Pi may combine, or with separate activator and inhibitor sites which cannot be filled simultaneously. Comparative initial velocity and binding studies with Pi indicate that the initial rate of AMP hydrolysis depends primarily on the extent of modifier site saturation with MgATP2-. Thus when two sites are filled with MgATP2-, the initial rate is approximately the same as when two additional modifier sites are filled with Pi. Binding of Pi, therefore, does not appear to affect the catalytic effectiveness of the active site when MgATP2- is also present, except by the displacement of MgATP2-.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D001395:Azotobacter / Q000201:enzymology; D001665:Binding Sites; D003956:Dialysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D014372:Tubercidin; D014462:Ultrafiltration
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D009699:N-Glycosyl Hydrolases; D008274:Magnesium; D014372:Tubercidin