Regulation of an Escherichia coli/mammalian chimeric carbamoyl-phosphate synthetase.
Carbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5'-pyrophosphate (PRPP) with a dissociation constant of 25 microM. The dissociation constant for UTP of 0.23 mM was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding.
Sahay
N
N
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Guy
H I
HI
Liu
X
X
Evans
D R
DR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics / Q000378:metabolism*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D008024:Ligands; D008322:Mammals / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion
D001639:Bicarbonates; D008024:Ligands; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D002221:Carbamyl Phosphate; D010754:Phosphoribosyl Pyrophosphate; D000641:Ammonia; D000255:Adenosine Triphosphate; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)