Nucleotide recognition in the ATP-grasp protein carbamoyl phosphate synthetase.
Synthesis of carbamoyl phosphate by carbamoyl phosphate synthetase (CPS) requires the coordinated utilization of two molecules of ATP per reaction cycle on duplicated nucleotide-binding sites (N and C). To clarify the contributions of sites N and C to the overall reaction, we carried out site-directed mutagenesis aimed at changing the substrate specificity of either of the two sites from ATP to GTP. Mutant design was based in part on an analysis of the nucleotide-binding sites of succinyl-CoA synthetases, which share membership in the ATP-grasp family with CPS and occur as GTP- and ATP-specific isoforms. We constructed and analyzed Escherichia coli CPS single mutations A144Q, D207A, D207N, S209A, I211S, P690Q, D753A, D753N, and F755A, as well as combinations thereof. All of the mutants retained ATP specificity, arguing for a lack of plasticity of the ATP sites of CPS with respect to nucleotide recognition. GTP-specific ATP-grasp proteins appear to accommodate this substrate by a displacement of the base relative to the ATP-bound state, an interaction that is precluded by the architecture of the potassium-binding loop in CPS. Analysis of the ATP-dependent kinetic parameters revealed that mutation of several residues conserved in ATP-grasp proteins and CPSs had surprisingly small effects, whereas constructs containing either A144Q or P690Q exerted the strongest effects on ATP utilization. We propose that these mutations affect proper movement of the lids covering the active sites of CPS, and interfere with access of substrate.
Kothe
Michael
M
Department of Biology, Northeastern University, Boston, MA 02115, USA.
Powers-Lee
Susan G
SG
D000225:Adenine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry*; D010856:Columbidae; D017124:Conserved Sequence / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine
D002352:Carrier Proteins; D008565:Membrane Proteins; C410230:tamalin protein, human; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D000225:Adenine