Asymmetric allosteric signaling in aspartate transcarbamoylase.
Here we use the fluorescence from a genetically encoded unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (HCE-Gly), replacing an amino acid in the regulatory site of Escherichia coli aspartate transcarbamoylase (ATCase) to decipher the molecular details of regulation of this allosteric enzyme. The fluorescence of HCE-Gly is exquisitely sensitive to the binding of all four nucleotide effectors. Although ATP and CTP are primarily responsible for influencing enzyme activity, the results of our fluorescent binding studies indicate that UTP and GTP bind with similar affinities, suggesting a dissociation between nucleotide binding and control of enzyme activity. Furthermore, while CTP is the strongest regulator of enzyme activity, it binds selectively to only a fraction of regulatory sites, allowing UTP to effectively fill the residual ones. Our results suggest that CTP and UTP are not competing for the same binding sites, but instead reveal an asymmetry between the two allosteric sites on the regulatory subunit of the enzyme. Correlation of binding and activity measurements explain how ATCase uses asymmetric allosteric sites to achieve regulatory sensitivity over a broad range of heterotropic effector concentrations.
Mendes
Kimberly R
KR
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Martinez
Jessica A
JA
Kantrowitz
Evan R
ER
D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011685:Purine Nucleotides / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D011685:Purine Nucleotides; D003570:Cytidine Triphosphate; D001221:Aspartate Carbamoyltransferase