Glutamine domain of the chimeric protein, CAD, that initiates pyrimidine biosynthesis in mammalian cells

Conference · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5333665

Kelly, R E; Kim, H; Evans, D R

Glutamine dependent carbamyl phosphate synthesis, the first step in mammalian de novo pyrimidine biosynthesis, is catalyzed by a 240 kDa chimeric protein, CAD, that also has the aspartate transcarbamylase and dihydroorotase activities. The complex was found to have a separate glutaminase activity of 0.04 ..mu..mol/min/mg, that increased five fold in the presence of bicarbonate and ATP. To determine whether the glutaminase activity, which provides ammonia for carbamyl phosphate synthesis, is associated with a separate structural domain (GLN), CAD was subjected to controlled proteolysis with elastase. The glutaminase, glutamine and ammonia dependent carbamyl phosphate synthetase activities, as well as the partial reactions; carbamyl phosphate dependent ATP synthesis and bicarbonate dependent ATPase, were correlated with the concentration of the various proteolytic fragments that accumulated in the digest. While the glutamine dependent carbamyl phosphate synthetase was rapidly inactivated, the glutaminase activity was found to be very resistant to proteolysis. The glutamine binding site of CAD was also specifically modified with 6-diazo-5-oxo-L-norleucine (DON). The modification was accompanied by a loss of both glutaminase and glutamine dependent carbamyl phosphate synthetase activities. Bicarbonate and ATP increased the rate of reaction of CAD with DON, while glutamine protected against inactivation. The stoichiometry of the reaction and the identity of the modified proteolytic fragments was determined using /sup 14/C labelled DON.

• Research Organization: Wayne State Univ. School of Medicine, Detroit, MI
• OSTI ID: 5333665
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AMIDASES
AMIDES
AMINO ACIDS
ATP-ASE
AZINES
BIOCHEMICAL REACTION KINETICS
BIOSYNTHESIS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
ENZYME ACTIVITY
ENZYMES
GLUTAMINE
HETEROCYCLIC COMPOUNDS
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIGASES
MEMBRANE PROTEINS
NON-PEPTIDE C-N HYDROLASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHOHYDROLASES
PROTEINS
PROTEOLYSIS
PYRIMIDINES
REACTION KINETICS
RECEPTORS
SERINE PROTEINASES
STOICHIOMETRY
SYNTHESIS
TRACER TECHNIQUES