Essential histidine residues of ribulosebisphosphate carboxylase indicated by reaction with diethylpyrocarbonate and rose bengal

Abstract

Modification of ribulosebisphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) by diethylpyrocarbonate or rose bengal-sensitized photooxidation caused rapid inactivation of the enzyme. The photooxidation proceeded following pseudo-first-order reaction kinetics showing a maximal value at pH 8.0. The fully activated enzyme was more sensitive to photooxidation as compared to the unactivated enzyme. The enzyme partially inactivated by photooxidation was fully sensitive to the positive effectors. The photooxidised enzyme showed a characteristic increase in absorbance at 250 nm which was dependent on the extent of inactivation. The kinetic analyses and correlation of the spectral changes with the activity indicated that the inactivation by diethylpyrocarbonate resulted from the modification of an average one histidine residue/70 000-dalton combination of large and small subunit. Sulfhydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. Ribulosebisphosphate and some effectors of the enzyme offered significant protection against diethylpyrocarbonate modification indicating that diethylpyrocarbonate was interacting with the enzyme at or near the active site.