Two and 8-azido photoaffinity probes. 2. Studies on the binding process of 2-5A synthetase by photosensitive ATP analogues
The photoaffinity probes (..gamma..-/sup 32/P)2-azidoATP (2-N/sub 3/ATP) and (..cap alpha..-/sup 32/P)8-azido-ATP (8-N/sub 3/ATP) were used to investigate the binding of ATP to highly purified 2-5A synthetase. 2-N/sub 3/APT and 8-N/sub 3/ATP are substrates for 2-5A synthetase. In this study the authors show that 2- and 8-N/sub 3/ATP are competitive inhibitors of the enzymatic conversion of ATP to 2-5A. Ultraviolet irradiation results in the photoinsertion of 2-N/sub 3/ATP and 8-N/sub 3/ATP into the enzyme. The covalent photoinsertion of (..cap alpha..-/sup 32/P)8-N/sub 3/ATP 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-N/sub 3/ATP and 2.0 mM 8-N/sub 3/ATP. Computer analysis of the curvilinear Scatchard plots of the 2-5A synthetase suggest 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-N/sub 3/ATP into the binding site(s) of the synthetase was determined. Photoinsertion of 8-N/sub 3/ATP into 2-5A synthetase increases with the addition of poly(rI)/times/poly(rC). Without the addition of poly(rI)/times/poly(rC) to the synthetase, the (..cap alpha..-/sup 32/P)8-N/sub 3/ATP is photoinserted into the enzyme; however, in the absence of dsRNA and in the absence of UV irradiation, the synthetase cannot convert ATP to 2-5A. The finding suggest that the formation of the enzyme/substrate complex can occur in the absence of dsRNA but dsRNA is essential to activate the 2-5A synthetase to form the productive complex needed for synthesis of 2-5A from ATP.
- Research Organization:
- Temple Univ. School of Medicine, Philadelphia, PA (USA)
- OSTI ID:
- 6208669
- Journal Information:
- Biochemistry; (United States), Vol. 27:24
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP
BIOLOGICAL RADIATION EFFECTS
CONFIGURATION INTERACTION
AZIDO COMPOUNDS
BIOCHEMISTRY
LIGASES
ENZYME ACTIVITY
ENZYME INHIBITORS
EXTREME ULTRAVIOLET RADIATION
PHOSPHORUS 32
RNA-ASE
SUBSTRATES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
CHEMISTRY
DAYS LIVING RADIOISOTOPES
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERASES
HYDROLASES
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LIGHT NUCLEI
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS ISOTOPES
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
ULTRAVIOLET RADIATION
550201* - Biochemistry- Tracer Techniques