Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states.
The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment conditions, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: (a) The role of GMP is that of an obligatory allosteric regulator. (b) Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. (c) GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, ethylenediamine-tetraacetic acid (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. (d) Formation of a GDP/guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. The results suggest that covalent modification or nucleotide analogue occupancy of the regulatory complex can modify the allosteric role for GMP, with consequences for the rate of the slow step.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology; D019204:GTP-Binding Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D011956:Receptors, Cell Surface / Q000378:metabolism; D013329:Structure-Activity Relationship; D014422:Turkeys
D008024:Ligands; D011956:Receptors, Cell Surface; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D006157:Guanosine Monophosphate; D004492:Edetic Acid; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol