title: The influence of quaternary structure on the active site of an oligomeric enzyme. Catalytic subunit of aspartate transcarbamoylase.
abstract: The catalytic subunit of aspartate transcarbamoylase from Escherichia coli reacts readily with 2,4,6-trinitrobenzenesulfonate, resulting in the loss of enzymatic activity. Substrates and substrate analogs protect the enzyme in a competitive manner, indicating that the loss of activity is due to modification of active-site residues. This conclusion was confirmed by fractionating tryptic digests of the modified protein followed by the identification of active-site lysines 83 and 84 as the modified residues. When three trinitrophenyl groups are incorporated per catalytic trimer, 70% of the activity is lost. The modified protein retains the sedimentation velocity and electrophoretic properties of the native catalytic subunit and can associate with regulatory subunit to form a holoenzyme-like molecule. The trinitrophenylated catalytic trimers have two strong absorption bands at 345 and 420 nm which serve as sensitive spectral probes in difference-spectroscopy experiments. Results from such experiments show that 1) the modified trimeric enzyme binds active-site ligands; 2) dissociation of the trimer into compact, highly structured monomers gives a spectral response distinguishable from that observed when the chains are completely unfolded; and 3) even though dissociation of the trimers to folded monomers causes the complete loss of enzyme activity, the resulting monomers still retain the ability to bind the bisubstrate analog N-(phosphonacetyl)-L-aspartate. These results indicate that the active site must be at least partially formed in the absence of any quaternary structure.
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000032:analysis*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D013053:Spectrophotometry; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology
chemical_list: D046911:Macromolecular Substances; D001224:Aspartic Acid; C013195:sparfosic acid; D014302:Trinitrobenzenesulfonic Acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid