Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5 prime -(p-(fluorosulfonyl)benzoyl)adenosine
- Wayne State Univ., Detroit, MI (United States)
- Univ. of Windsor, Ontario (Canada)
The ATP analogue 5{prime}-(p-(fluorosulfonyl)benzoyl)adenosine (FSBA) was used to chemically modify the ATP binding sites of the carbamyl phosphate synthetase domain of CAD, the multifunctional protein that catalyzes the first steps in mammalian pyrimidine biosynthesis. Reaction of CAD with FSBA resulted in the inactivation of the ammonia- and glutamine-dependent CPSase activities but had no effect on its glutaminase, aspartate transcarbamylase, or dihydroortase activities. ATP protected CAD against inactivation by FSBA whereas the presence of the allosteric effectors UTP and PRPP afforded little protection, which suggests that the ATP binding sites were specifically labeled. The inactivation exhibited saturation behavior with respect to FSBA with a K{sub I} of 0.93 mM. The stoichiometry of ({sup 14}C)FSBA labeling showed that only 0.4-0.5 mol of FSBA/mol of protein was required for complete inactivation. Incorporation of radiolabeled FSBA into CAD and subsequent proteolysis, gel electrophoresis, and fluorography demonstrated that only the carbamyl phosphate synthetase domain of CAD is labeled. Amino acid sequencing of the principal peaks resulting from tryptic digests of FSBA-modified CAD located the sites of FSBA modification in regions that exhibit high homology to ATP binding sites of other known proteins. Thus CAD has two ATP binding sites, one in each of the two highly homologous halves of the carbamyl phosphate domain which catalyze distinct ATP-dependent partial reactions in carbamyl phosphate synthesis.
- OSTI ID:
- 5687474
- Journal Information:
- Biochemistry; (United States), Vol. 30:42; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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LIGASES
AMINO ACID SEQUENCE
NUCLEOSIDES
AUTORADIOGRAPHY
ACID CARBONATES
ADENOSINE
ATP
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
ESCHERICHIA COLI
STOICHIOMETRY
BACTERIA
CARBON COMPOUNDS
ENZYMES
KINETICS
LABELLED COMPOUNDS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEOTIDES
ORGANIC COMPOUNDS
PROTEINS
REACTION KINETICS
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550201* - Biochemistry- Tracer Techniques