On the role of substrate and GTP in the regulation of argininosuccinase activity.
As determined by equilibrium dialysis, bovine liver argininosuccinase of molecular weight 202,000 binds 4 mol of argininosuccinate or arginine/mol of enzyme. Negative homotropic interactions occur in the binding of both ligands at 0.15 ionic strength in the presence of phosphate. Argininosuccinate binds to two sites (Kdiss 1.6 times 10(-5) M) and four sites (Kdiss 2.9 times 10(-4) M) at low and high substrate concentration. Similarly, arginine binds to two sites (Kdiss 4.9 times 10(-4) M), and four sites (Kdiss 1.6 times 10(-3) M). At 0.05 ionic strength in Tris-HCl buffer, the four enzyme sites bind argininosuccinate independently and arginine binding remains negatively cooperative. Kinetic analysis gave double reciprocal plots that showed negative cooperatively also. The changes in Km were analogous to changes in Kdiss, thus indicating that the substrate binding sites correspond to catalytic sites. Since the catalytically active enzyme is a tetramer composed of four identical or closely similar subunits (Lusty, C.J., and Ratner, S. (1972) J. Biol. Chem. 247, 7010-7022), the present results show that each subunit contains one catalytic site. Ionic strength, phosphate ions, and GTP have each been found to influence negative cooperatively through a change in the affinity for argininosuccinate. The significance of the negative homotropic interactions and of the specific stimulation of activity by GTP is discussed with respect to different conformational forms of the enzyme and the in vivo regulation of argininosuccinase activity.
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001123:Argininosuccinate Lyase / Q000378:metabolism*; D001125:Argininosuccinic Acid / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008190:Lyases / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009994:Osmolar Concentration; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D014325:Tromethamine / Q000494:pharmacology
D046911:Macromolecular Substances; D010710:Phosphates; D014325:Tromethamine; D001125:Argininosuccinic Acid; D006160:Guanosine Triphosphate; D001120:Arginine; D008190:Lyases; D001123:Argininosuccinate Lyase