Studies of the thermal inactivation of cardiac adenylyl cyclase: evidence for a conformational change in the reaction mechanism.
Membrane bound cardiac adenylyl cyclase was shown to undergo a spontaneous and irreversible thermal inactivation with a t1/2 of approximately 10 min. The loss of activity could not be explained by the action of endogenous proteases. Repeated freeze-thaw of membrane preparations resulted in a much increased rate of thermal inactivation (t1/2 = approx. 2 min). ATP, adenylimidodiphosphate, ADP, and PPi protected the enzyme from thermal inactivation with dissociation constants (Kd) of 193, 5.04, 84.4, and 6.3 microM, respectively. 5'-AMP and cyclic AMP were ineffective as protectors at concentrations as high as 3 mM. Activators of adenylyl cyclase such as Mn2+, forskolin, 5-guanylylimidodiphosphate, and NaF and 9 mM Mg2+ protected against thermal inactivation with Kd of 16.8 microM, 8.81 microM, 0.23 microM and 1.04 mM, respectively. Mg2+ alone was without effect. Thermal inactivation was first order under all conditions tested. Arrhenius plots of the rate constants for inactivation vs temperature were linear. The increased stability of ligand bound adenylyl cyclase was shown to be associated with an increased free energy of activation (delta G 0). These data provide evidence for the existence of two distinct conformations of cardiac adenylyl cyclase based on different susceptibilities to thermal inactivation. These enzyme conformations, termed E1 and E2, may be important reaction intermediates. The thermal stability of E1 was highly influenced by the enzyme's membrane lipid environment. The formation of E2 from E1 was enhanced by interaction with substrate, PPi, activators of adenylyl cyclase, and by interaction with dissociated stimulatory guanine nucleotide binding protein-alpha beta gamma heterotrimers.
Colvin
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Department of Zoological and Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens 45701.
Allen
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RA
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D004285:Dogs; D005615:Freezing; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D012508:Sarcolemma / Q000201:enzymology; D013696:Temperature; D013816:Thermodynamics
D000067956:Adenylyl Cyclase Inhibitors; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; D008274:Magnesium