2- and 8-azido photoaffinity probes. 2. Studies on the binding process of 2-5A synthetase by photosensitive ATP analogues.
The photoaffinity probes [gamma-32P]2-azidoATP (2-N3ATP) and [alpha-32P]8-azido-ATP (8-N3ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N3ATP and 8-N3ATP are substrates for 2-5A synthetase [Suhadolnik, R.J., Karikó, K., Sobol, R.W., Jr., Li, S.W., Reichenbach, N.L., & Haley, B.E., preceding paper]. In this study we show that 2- and 8-N3ATP are competitive inhibitors of the enzymatic conversion of ATP to 2-5A. Ultraviolet irradiation results in the photoinsertion of 2-N3ATP and 8-N3ATP into the enzyme. The covalent photoinsertion of [alpha-32P]8-N3ATP into the 2-5A synthetase is proportional to the inactivation of the enzyme as UV irradiation is increased. Photolabeling of 2-5A synthetase is saturated at 1.5 mM 2-N3ATP and 2.0 mM 8-N3ATP. Computer analysis of the curvilinear Scatchard plots of the 2-5A synthetase suggests the presence of high-affinity and low-affinity binding sites that may correspond to the acceptor and the 2'-adenylation sites of the enzyme. The competition of nucleotides for the covalent photoinsertion of 8-N3ATP into the binding site(s) of the synthetase was as follows: ATP greater than 2'dATP = 3'dATP greater than CTP greater than ITP greater than AMP greater than NAD+ greater than UTP greater than UMP greater than CMP. Photoinsertion of 8-N3ATP into 2-5A synthetase increases with the addition of poly(rI).poly(rC).(ABSTRACT TRUNCATED AT 250 WORDS)
Suhadolnik
R J
RJ
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Li
S W
SW
Sobol
R W
RW
Haley
B E
BE
D015088:2',5'-Oligoadenylate Synthetase / Q000378:metabolism* / Q000528:radiation effects; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D001667:Binding, Competitive; D011756:Diphosphates / Q000032:analysis; D007700:Kinetics; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D011756:Diphosphates; D010761:Phosphorus Radioisotopes; C017040:8-azidoadenosine 5'-triphosphate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D015088:2',5'-Oligoadenylate Synthetase