Binding of regulatory nucleotides to aspartate transcarbamylase: nuclear magnetic resonance studies of selectively enriched carbon-13 regulatory subunit

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Abstract
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 3 tyrosines and 4 histidines per regulatory chain give narrow, well resolved resonances, and the signals from the 5 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 2 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 to that observed for isolated regulatory subunit. This histidine spectrum was undisturbed by the T to R conformational transition of the enzyme. Apparently, the regulatory subunit experiences minimal rearrangement of tertiary structure on binding effectors and at least 1 phenylalanine and 1 histidine residue are present in the region of the CTP binding site.