Gain of glutaminase function in mutants of the ammonia-specific frog carbamoyl phosphate synthetase.
Depending on their physiological role, carbamoyl phosphate synthetases (CPSs) use either glutamine or free ammonia as the nitrogen donor for carbamoyl phosphate synthesis. Sequence analysis of known CPSs indicates that, regardless of whether they are ammonia- or glutamine-specific, all CPSs contain the structural equivalent of a triad-type glutamine amidotransferase (GAT) domain. In ammonia-specific CPSs, such as those of rat or human, the catalytic inactivity of the GAT domain can be rationalized by the substitution of the Triad cysteine residue by serine (1). The ammonia-specific CPS of Rana catesbeiana (fCPS) presents an interesting anomaly in that, despite its retention of the entire catalytic triad (2) and almost all other residues conserved in Triad GATs, it is unable to utilize glutamine as a nitrogen-donating substrate (3). Based on our earlier work with the glutamine-utilizing E. coli CPS (eCPS), we have targeted residues Lys258 and Glu261 in the fCPS GAT domain as critical for preventing GAT function. Previously we have shown that substitution of the corresponding residues in eCPS by their fCPS counterparts (Leu --> Lys and Gln --> Glu) resulted in complete loss of GAT function in eCPS (3). To examine the role of these residues in the fCPS GAT component, we have cloned the full-length fCPS gene from R. catesbeiana liver. Here we report the first heterologous expression of an ammonia-specific CPS and show that a single mutation of the frog enzyme, K258L, yields a gain of glutaminase function.
Saeed-Kothe
Amna
A
Department of Biology, Northeastern University, Boston, Massachussetts 02115, USA.
Powers-Lee
Susan G
SG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D005972:Glutaminase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011892:Rana catesbeiana / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D005972:Glutaminase; D002222:Carbamoyl-Phosphate Synthase (Ammonia)