Two and 8-azido photoaffinity probes. 2. Studies on the binding process of 2-5A synthetase by photosensitive ATP analogues

Journal Article · Mon Nov 28 23:00:00 EST 1988 · Biochemistry; (United States)

OSTI ID:6208669

Suhadolnik, R J; Li, S W; Sobol, Jr, R W; Haley, B E

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), Journal Name: Biochemistry; (United States) Vol. 27:24; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ATP
AZIDO COMPOUNDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CHEMISTRY
CONFIGURATION INTERACTION
DAYS LIVING RADIOISOTOPES
ELECTROMAGNETIC RADIATION
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
ESTERASES
EXTREME ULTRAVIOLET RADIATION
HYDROLASES
ISOTOPES
LIGASES
LIGHT NUCLEI
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
RNA-ASE
SUBSTRATES
ULTRAVIOLET RADIATION