Activation of adenylate cyclase by phosphoramidate and phosphonate analogs of GTP: possible role of covalent enzyme-substrate intermediates in the mechanism of hormonal activation.
Incubation of rat fat pad membranes with 5-guanylyliminodiphosphonate [Gpp-(NH)p] and 5-guanylylmethylenediphosphonate [Gpp(CH2)p], but not GTP (with or without hormones), at 24 degrees or 30 degrees (but not at 4 degrees) greatly stimulates adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] measured after thoroughly washing the membranes. The rate of activation is relatively slow, even with very high (and saturating) concentrations of the analogs. Binding alone appears to be insufficient for activation. Hormones (catecholamines, glucagon) increase the rate but not the extent of activation, even when saturating analog concentrations are used. The dependence on analog concentration (apparent Km) varies with the time of incubation. GTP and very high concentrations of ATP inhibit the activation by Gpp(NH)p, but this effect is dependent on the length of incubation and can be overcome with time. The activated state is not reversed upon incubation of the washed membranes at 30 degrees, even in the presence of GTP, or by solubilization with nonionic detergents. Also, Gpp(NH)p can directly stimulate the control, solubilized enzyme. The activated state of the solubilized enzyme persists upon specific adsorption to and subsequent elution from an organomercurial-agarose column. It is suggested that after forming reversible Michaelis complexes of relatively low affinity, these analogs may react irreversibly with the GTP regulatory site of the enzyme, perhaps forming p(NH)p- and p(CH2)p-covalent enzyme intermediates which capture the activated state of the enzyme. GTP, after binding, may normally activate the enzyme by forming a "labile" pyrophosphoryl enzyme intermediate, and hormone receptors may function to increase the rate of formation (and thus concentration) of this active state of the enzyme.
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000273:Adipose Tissue / Q000187:drug effects / Q000201:enzymology*; D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002846:Chromatography, Affinity; D004789:Enzyme Activation / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D005934:Glucagon / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D063065:Organophosphonates / Q000494:pharmacology*; D051381:Rats
D000577:Amides; D063065:Organophosphonates; D006160:Guanosine Triphosphate; D005934:Glucagon; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D005459:Fluorides