Modification of an essential amino group of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal phosphate and by pyridoxal phosphate-sensitized photooxidation.

Phosphoenolpyruvate carboxylase from maize leaves was inactivated by pyridoxal 5'-phosphate in the dark and in the light. A two-step reversible mechanism is proposed for inactivation in the dark, which involves the formation of a noncovalent complex prior to a Schiff base with amino groups of the enzyme. Spectral analysis of pyridoxal 5'-phosphate-modified phosphoenolpyruvate carboxylase showed absorption maxima at 432 and 327 nm, before and after reduction with NaBH4, respectively, suggesting that epsilon-amino groups of lysine residues are the reactive groups in the enzyme. A correlation between spectral data and the maximal inactivation obtained with several concentrations of inhibitor allowed us to establish that the incorporation of 4 mol of pyridoxal 5'-phosphate per mole of holoenzyme accounts for total inactivation. The absence of modifier bound to phosphoenolpyruvate carboxylase when the modification was carried out in the presence of phosphoenolpyruvate and MgCl2 suggests the existence of an essential lysine residue at the catalytic site of the enzyme. Modification of phosphoenolpyruvate carboxylase in the light under an oxygen atmosphere resulted in an irreversible inactivation, which was completely protected by phosphoenolpyruvate and MgCl2. Spectral analysis of the photomodified enzyme showed an absorption peak of 320 nm, suggesting light-mediated addition of a nucleophilic residue (probably an imidazole group) to the pyridoxal 5'-phosphate-lysine azomethine bond.