An Essential Histidine Residue in the Catalytic Mechanism of the Rat Kidney gamma-Glutamyl Transpeptidase

摘要

gamma-Glutamyl transpeptidase (EC 2.3.2.2) plays a key role in glutathione metabolism by catalyzing the transfer of the gamma-glutamyl residue and hydrolysis of glutathione.The functional residues at the active site of the rat kidney gamma-glutamyl transpeptidase were investigated by kinetic studies at various pH,the treatment of diethylpyrocarbonate (DEPC),and photooxidation in presence of methylene blue.An ionizable group affecting the enzymatic activity with an apparent pK_a value of 7.1,which is in the range of pK_a.values for a histidine residue in protein,was obtained by examining the pH-dependence of kinetic parameters.The pH effect on the photoinduced inactivation rate of the enzyme corresponds to that expected for the photooxidation of the free histidine.The involvement of a histidine in the catalytic site of the enzyme was further supported by DEPC modification accompanied by an increase in absorbance at 240 nm,indicating the formation of N-carbethoxyhistidine.The histidine located at the position of 382 in the precursor of the enzyme is primarily suspected based on the amino acid sequence alignment of the transpeptidases from various organisms.