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Photoaffinity labeling of rat liver carbamoyl phosphate synthetase I by 8-azido-ATP

Journal Article · · J. Biol. Chem.; (United States)
OSTI ID:6170312
;

8-Azido-ATP has been found to serve as a photoaffinity label for two distinct ATP sites on rat liver carbamoyl phosphate synthetase I and to allow preliminary localization of these sites. In the dark, 8-azido-ATP acted as a competitive inhibitor with respect to ATP. Ultraviolet irradiation of carbamoyl phosphate synthetase I in the presence of 8-azido-ATP led to an irreversible loss of activity. ATP specifically protected against this inactivation. The incorporation of 2 mol of 8-azido-ATP per mol of enzyme was required for complete inactivation. To localize the 8-azido-ATP-binding sites to discrete regions of carbamoyl phosphate synthetase I which appear to be structural domains, the enzyme was photolabeled with (gamma-/sup 32/P)8-azido-ATP and subjected to limited proteolytic digestion. The resulting model for the functional roles of the domains is that there is one ATP site on each of the two large internal structural domains of the enzyme. Each of these domains was found to contain the consensus sequences A and B common to many other nucleotide-binding proteins. In addition, there is extensive structural and possibly functional interaction of the smaller N-terminal domain with one of the internal ATP-binding domains, analogous to a subunit interaction observed with the evolutionarily related Escherichia coli carbamoyl phosphate synthetase.

Research Organization:
Northeastern Univ., Boston, MA
OSTI ID:
6170312
Journal Information:
J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 262:19; ISSN JBCHA
Country of Publication:
United States
Language:
English

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Related Subjects

560120* -- Radiation Effects on Biochemicals
Cells
& Tissue Culture
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACID SEQUENCE
ANIMALS
ATP
AZIDES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BODY
CHEMISTRY
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTROMAGNETIC RADIATION
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
GLANDS
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LIGASES
LIGHT NUCLEI
LIVER
MAMMALS
MEMBRANE PROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NITROGEN COMPOUNDS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOTOCHEMISTRY
PROTEINS
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
RADIOSENSITIVITY EFFECTS
RATS
RECEPTORS
RODENTS
TRACER TECHNIQUES
ULTRAVIOLET RADIATION
VERTEBRATES