Binding of regulatory nucleotides to aspartate transcarbamylase: nuclear magnetic resonance studies of selectively enriched carbon-13 regulatory subunit.
Specifically enriched [gamma-13C]phenylalanine, -tyrosine, and -histidine have been biosynthetically incorporated into aspartate transcarbamylase from Escherichia coli. These nonperturbing NMR probes have been used to characterize the interaction of the regulatory sites on the enzyme with nucleotide effectors. The C gamma carbons of the three tyrosines and four histidines per regulatory chain give narrow, well-resolved resonances, and the signals from the five phenylalanines per chain are partially resolved in the presence of bound inhibitor. Spectral changes in regulatory subunit were monitored as a function of concentration of the inhibitor, CTP, and the activator, ATP. Three histidine residues responded to ATP and CTP in an identical manner while two phenylalanine residues were sensitive to CTP but not ATP binding. The tyrosine resonances were not perturbed by effectors. The chemical shift response of the single observable histidine resonance to bound nucleotides in the reconstituted enzyme was identical with that observed for isolated regulatory subunit. This histidine spectrum was undisturbed by the T to R conformational transition of the enzyme. The results suggest that the regulatory subunit experiences minimal rearrangement of tertiary structure on binding effectors and that at least one phenylalanine and one histidine residue are present in the region of the CTP binding site.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000494:pharmacology*; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D010649:Phenylalanine; D011485:Protein Binding; D014443:Tyrosine
D003597:Cytosine Nucleotides; D046911:Macromolecular Substances; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase