Aspartate transcarbamylase from Escherichia coli. The use of pyridoxal 5'-phosphate as a probe in the active site.

Spectrophotometric titration studies at pH 8.0 have revealed that pyridoxal 5’-phosphate binds as a Schiff base to the catalytic subunit of Escherichia coli aspartate transcarbamylase with a stoichiometry of 3 pyridoxal phosphates per trimer and a dissociation constant of 0.9 pM. The ultraviolet absorption of the Schiff base near 430 nm is lost upon addition of the inhibitor N-(phosphonacetyl)-L-aspartate (PALA) or the substrate carbamyl-P. In steady state kinetic studies, pyridoxal-P was found to be a competitive inhibitor of carbamyl-P, with an inhibition constant of 4 f 2 PM. Pyridoxal does not bind to or inhibit the catalytic subunit. These results imply that pyridoxal-P binds at the active site not only through a Schiff base but also through noncovalent, ionic interactions with the phosphate binding region. Reduction of the Schiff base with sodium borohydride yields an N-E-pyridoxyl-L-lysine derivative of the enzyme with 2.9 to 3.0 pyridoxal-P incorporated per catalytic trimer. This reduced derivative is inactive but still binds 3 molecules of PALA per catalytic trimer at pH 8.0. However, the binding of PALA is characterized by at least two dissociation constants in the range 100 to 10 PM. PALA binds much more tightly to unmodified catalytic subunit at pH 8 with a single dissociation constant of about 0.1 PM. Photo-oxidation of the unreduced catalytic subunit-pyridoxal-P Schiff base causes loss of 90% to 95% of the activity and loss of 2.0 f 0.5 histidine residues per chain. No other amino acid is found to be modified by amino acid analysis. However, 0.5 f 0.05 molecules per chain of an unknown derivative of pyridoxal-P derivative are covalently bound. Covalently linked dimers and trimers of the catalytic chains are also formed, especially at high protein concentrations. At low concentrations at which dimer and trimer formation is negligible, the covalent incorporation of 0.5 pyridoxal-P per chain does not account for the almost complete loss of enzyme activity. Therefore, the loss of histidine is correlated with loss of activity. These studies indicate that 1 lysine residue and 2 histidine residues are probably near the active site of aspartate transcarbamylase.