Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain
- Instituto de Investigaciones Citologicas de la Caja de Ahorros de Valencia (Spain)
- The Public Health Research Institute, New York, NY (USA)
- St. Mary's Hospital Medical School, London (England)
The large subunit of Escherichia coli carbamoyl phosphate synthetase is responsible for carbamoyl phosphate synthesis from NH{sub 3} and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of ({sup 14}C)UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradation with ({sup 14}C)UMP. The ({sup 14}C)UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. Since the binding sites for IMP and UMP overlap, most probably IMP also binds in this domain. The protection from proteolysis by ornithine suggests that ornithine binds in the same domain. To account for the effects of the allosteric effectors on the binding of ATP, the authors propose a scheme where the two halves of the large subunit form a pseudohomodimer by complementary isologous association, thus placing the NH{sub 2} half, which is involved in the binding of the molecule of ATP that yields P{sub i}, close to the regulatory domain.
- OSTI ID:
- 5398972
- Journal Information:
- Biochemistry; (United States), Vol. 30:4; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ENZYME INHIBITORS
BIOLOGICAL RADIATION EFFECTS
ESCHERICHIA COLI
BIOLOGICAL PATHWAYS
LIGASES
INACTIVATION
ARGININE
CARBAMIC ACID ESTERS
CARBON 14 COMPOUNDS
ORNITHINE
PYRIMIDINES
ULTRAVIOLET RADIATION
UREA
AMIDES
AMINO ACIDS
AZINES
BACTERIA
BIOLOGICAL EFFECTS
CARBON COMPOUNDS
CARBONIC ACID DERIVATIVES
CARBOXYLIC ACID ESTERS
CARBOXYLIC ACIDS
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERS
HETEROCYCLIC COMPOUNDS
LABELLED COMPOUNDS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
RADIATION EFFECTS
RADIATIONS
550201* - Biochemistry- Tracer Techniques