The synthesis of carbamyl phosphate, an intermediate in pyrimidine and arginine biosynthesis, is catalyzed by ammonia and glutamine dependent carbamyl phosphate synthetase (CPS). E coli has a single glutamine dependent CPS composed of a glutaminase (alpha subunit) and synthetase (Beta subunit). The two subunits are encoded by the carAB operon. The alpha subunit is a member of a large family of enzymes that provide amide nitrogen for diverse biosynthetic reactions. The Beta subunit probably evolved by duplication of the same ancestral gene that gave rise to carbamate synthetase. This enzyme has a complex mosaic structure with active sites related to the biotin containing carboxylases. Studies of carAB and site-directed mutants have enabled us to map the functional, regulatory, and subunit contact domains in the enzyme. The enzymatic and substrate binding properties of structurally native but catalytically impaired CPSs have allowed separate sequential steps of the reaction mechanism to be ascribed to two-nucleotide binding domains located separately in each duplicated half of the Beta subunit. The catalytic site mutants have also revealed reciprocal modulatory influences of active site domains located in the alpha and Beta subunits and have helped to identify some key residues underlying these interactions. These and earlier biochemical data of E coli CPS have contributed a good working model for more detailed probings of the structure-function relationships in the enzyme. Four objectives will be pursued during the coming period. Previous attempts to crystallize CPS have been sufficiently encouraging to warrant more intensified efforts along these lines. A major part of our time will be devoted to obtaining stable and highly diffracting crystals of CPS and/or of its constituent subunits. A collaborative arrangement has been established with Dr. Wayne Hendrickson's laboratory to solve the structure. We will also continue our studies of carB mutations affecting the partial reactions and active site coupling properties of the Beta subunit. These studies are anticipated to provide additional mechanistic details and identify catalytically important residues. The proposal that the catalytic mechanism of type G glutaminases is similar to that of cysteine proteinases has gained support from the demonstration of a thioester intermediate during the hydrolysis of glutamine and the observed sequence homology of the alpha subunit to papain. This model will be experimentally tested through studies of site-directed mutations in residues conserved among the two groups of proteins, particularly those residues known to affect the stability of cys-his ion pairing in papain. Finally, experiments are proposed to examine IMP and UMP induced quaternary conformational changes by the carboxyl proximal allosteric domain and of the resultant contribution by this domain of residues necessary for substrate and metal binding at the active sites of the Beta subunit.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025846-15
Application #
2174547
Study Section
Biochemistry Study Section (BIO)
Project Start
1980-04-01
Project End
1997-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
15
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Public Health Research Institute
Department
Type
DUNS #
City
Newark
State
NY
Country
United States
Zip Code
Cervera, J; Bendala, E; Britton, H G et al. (1996) Photoaffinity labeling with UMP of lysine 992 of carbamyl phosphate synthetase from Escherichia coli allows identification of the binding site for the pyrimidine inhibitor. Biochemistry 35:7247-55
Hong, J; Salo, W L; Lusty, C J et al. (1994) Carbamyl phosphate synthetase III, an evolutionary intermediate in the transition between glutamine-dependent and ammonia-dependent carbamyl phosphate synthetases. J Mol Biol 243:131-40
Bueso, J; Lusty, C J; Rubio, V (1994) Location of the binding site for the allosteric activator IMP in the COOH-terminal domain of Escherichia coli carbamyl phosphates synthetase. Biochem Biophys Res Commun 203:1083-9
Lusty, C J; Liao, M (1993) Substitution of Glu841 by lysine in the carbamate domain of carbamyl phosphate synthetase alters the catalytic properties of the glutaminase subunit. Biochemistry 32:1278-84
Cervera, J; Conejero-Lara, F; Ruiz-Sanz, J et al. (1993) The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry. J Biol Chem 268:12504-11
Kern, C B; Lusty, C J; Davidson, J N (1992) Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion. J Mol Evol 35:217-22
Guillou, F; Liao, M; Garcia-Espana, A et al. (1992) Mutational analysis of carbamyl phosphate synthetase. Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunit. Biochemistry 31:1656-64
Lusty, C J (1992) Detection of an enzyme bound gamma-glutamyl acyl ester of carbamyl phosphate synthetase of Escherichia coli. FEBS Lett 314:135-8
Mullins, L S; Lusty, C J; Raushel, F M (1991) Alterations in the energetics of the carbamoyl phosphate synthetase reaction by site-directed modification of the essential sulfhydryl group. J Biol Chem 266:8236-40
Rubio, V; Cervera, J; Lusty, C J et al. (1991) 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. Biochemistry 30:1068-75

Showing the most recent 10 out of 18 publications