Adenosine monophosphate nucleosidase (AMP nucleosidase, EC 126.96.36.199) has been purified Over 80-fold from Azotobacter vinelandii OP and used to determine the mechanism of inactivation which occuss when the enzyme is placed in a low ionic strength environment. The kinetics of inactivation, together with results of gel filtration studies, indicate that the native enzyme exists in a polymeric form with an approximate molecular weight of 360,000. The enzyme is maintained in this form by a variety of inorganic salts as well as by the substrate (AMP) or essential activator (MgATP2-). Removal of these protective agents results in a rapid loss of activity accompanied by the appearance of an inactive form of the enzyme having an apparent molecular weight of about 180,000. The inactive enzyme may be converted to the native form by the addition of salts, substrate, or activator. It is proposed that inorganic salts stabilize the native enzyme by neutralizing excess electrostatic charges in the molecule, which, when unshielded, disrupt the quaternary structure of the protein. Substrate and essential activator protect not by ionic shielding, but by combination at specific binding sites on the enzyme. Such combinations cause a conformational or charge distribution change which is sufficient to stabilize the quaternary structure in the absence of protection by inorganic salts. Comparison of initial velocity and stability experiments to both AMP and MgATP2- indicate that the substrate combines well with the enzyme either in the presence or absence of essential activator. Thus the function of MgATP2- must be to provide a catalytically active conformation rather than to form a binding site for the substrate.
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