Identification of essential histidine residues of aminoacylase by photooxidation and by reaction with diethylpyrocarbonate.

State and function of the histidine residues of aminoacylase were investigated by photoxidation in the presence of methylene blue and by chemical modification with diethylpyrocarbonate. Complete inactivation of the enzyme was observed after oxidation of 4 histidine residues. From the pH dependence of the photooxidation it becomes evident that the inactivation of the enzyme is not a consequence of the simultaneous oxidation of tryptophan residues. The enzyme is also inactivated by chemical modification of histidine residues with diethylpyrocarbonate. Activity is restored by treatment with hydroxylamine. Zn2+-ions which are essential for the activity of aminoacylase protect the available histidine molecules against photooxidation and attack by diethylpyrocarbonate. It is suggested that histidine is involved in the binding of the essential Zn2+-ions.