Covalent labeling of adenylyl cyclase cytosolic domains with gamma-methylimidazole-2',5'-dideoxy-[gamma-(32)P]3'-ATP and the mechanism for P-site-mediated inhibition.
A truncated first cytosolic domain of type V adenylyl cyclase (VC(1)) and a truncated second cytosolic domain of type II adenylyl cyclase (IIC(2)) were used alone and in the readily reversible complex (VC(1).IIC(2)) to evaluate interactions with each other and with reversible and irreversible P-site ligands. Enzyme activity was used to assess formation and dissolution of VC(1).IIC(2). The data suggest that binding of 2',5'-dideoxy-3'-ATP to VC(1) and IIC(2) prevented formation of VC(1).IIC(2) and that 2',5'-dideoxy-3'-ATP dissociation occurred slowly. To enable configuration specific cross-linking to the catalytic site, 2',5'-dideoxyadenosine 3'-[gamma-(1-methylimidazole)-triphosphate] (gamma-MetIm-2', 5'-dd-3'-ATP) and 2',5'-dd-adenosine 3'-(gamma-azidoanilido)-triphosphate (gamma-azidoanilido-2', 5'-dd-3'-ATP) were synthesized, the former also as its gamma-(32)P-labeled analog. gamma-Azidoanilido-2',5'-dd-3'-ATP exhibited an inhibitory potency comparable with that of 2', 5'-dd-3'-ATP. gamma-MetIm-2',5'-dd-[gamma-(32)P]3'-ATP labeled the individual VC(1) and IIC(2) domains comparably and covalently to approximately 20% within 1 h. Formation of VC(1).IIC(2) resulted in reduced labeling of VC(1) but enhanced labeling of IIC(2). The data imply that formation of the catalytically active VC(1).IIC(2) complex affects the interaction of each domain with the 2', 5'-dd-3'-ATP, the binding of which also affects the interaction between the two cytosolic domains, leading to a pseudo-irreversible inhibition.
Doronin
S
S
Department of Physiology, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8661, USA.
Murray
L
L
Dessauer
C W
CW
Johnson
R A
RA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D054306:Dideoxynucleotides; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007093:Imidazoles / Q000138:chemical synthesis / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D013997:Time Factors
C403310:2',5'-dideoxyadenosine 3'-(gamma-(1-methylimidazole)triphosphate); D000067956:Adenylyl Cyclase Inhibitors; D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D054306:Dideoxynucleotides; D007093:Imidazoles; D007527:Isoenzymes; C403309:gamma-azidoanilido-2',5'-dideoxyadenosine 3'-triphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases