Activation by fusion of the glutaminase and synthetase subunits of Escherichia coli carbamyl-phosphate synthetase.
Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.
Guy
H I
HI
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Rotgeri
A
A
Evans
D R
DR
D000255:Adenosine Triphosphate / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005972:Glutaminase / Q000378:metabolism; D008025:Ligases / Q000378:metabolism; D008970:Molecular Weight; D009952:Ornithine / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism
D001639:Bicarbonates; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D014542:Uridine Monophosphate; D009952:Ornithine; D010196:Pancreatic Elastase; D005972:Glutaminase; D008025:Ligases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)