O-phthalaldehyde as a probe in the active site of ribulose 1,5-bisphosphate carboxylase

Abstract

Ribulose-1,5-bisphosphate carboxylase (3-phospho- d-glycerate carboxy-lyase (dimerizing). EC 4.1.1.39) is rapidly and irreversibly inactivated by micromolar concentrations of o-phthalaldehyde in borate buffer. The inactivation followed pseudo-first-order reaction kinetics with a high second-order rate constant. The CO 2/Mg 2+-activated enzyme was more sensitive to o-phthalaldehyde inhibition as compared to the unactivated enzyme. The kinetic analysis and the correlation of the spectral changes with the carboxylase activity indicated that inactivation by o-phthalaldehyde resulted from modification of approximately one lysine and one cysteine residue per 67 kDa combination of large and small subunits. Several competitive inhibitors offered strong protection against o-phthalaldehyde inhibition. Studies with [ 3H]pyridoxal phos phate showed that both o-phthalaldehyde and pyridoxal phosphate react with the same lysine residue at the active site. Phthalaldehyde was incorporated in the large subunits, which resulted in the inter-linking of large subunits. The major cross-linked fraction was a dimer of the large subunit. It seems that o-phthalaldehyde introduced intersubunit cross-linking.