Modification of an Essential Amino Group of Phosphoenolpyruvate Carboxylase from Maize Leaves by Pyridoxal Phosphate and by Pyridoxal Phosphate Sensitized Photooxidation

F.E. PODESTÁ, A.A. IGLESIAS, C.S. ANDREO

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Año: 1986 vol. 246 p. 546 - 553

0003-9861

Phosphoenolpyruvate carboxylase from maize leaves was inactivated by pyridoxal5’-phosphate in the dark and in the light. A two-step reversible mechanism is proposedfor inactivation in the dark, which involves the formation of a noncovalent complexprior to a Schiff base with amino groups of the enzyme. Spectral analysis of pyridoxal5’-phosphate-modified phosphoenolpyruvate carboxylase showed absorption maxima at432 and 327 nm, before and after reduction with NaBH4, respectively, suggesting thatc-amino groups of lysine residues are the reactive groups in the enzyme. A correlationbetween spectral data and the maximal inactivation obtained with several concentrationsof 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 modifierbound to phosphoenolpyruvate carboxylase when the modification was carried out inthe presence of phosphoenolpyruvate and MgClz suggests the existence of an essentiallysine residue at the catalytic site of the enzyme. Modification of phosphoenolpyruvatecarboxylase in the light under an oxygen atmosphere resulted in an irreversible inactivation,which was completely protected by phosphoenolpyruvate and MgClz. Spectralanalysis of the photomodified enzyme showed an absorption peak at 320 nm, suggestinglight-mediated addition of a nucleophilic residue (probably an imidazole group) to thepyridoxal 5’-phosphate-lysine azomethine bond.