The influence of quaternary structure on the active site of an oligomeric enzyme. Catalytic subunit of aspartate transcarbamoylase.

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.