Abstract
Ethoxyformic anhydride and photooxidation were used to study the function of histidine residues in neutral proteinase from Streptomyces naraensis. Ethoxyformic anhydride completely inactivated the enzyme. However, NH2OH reactivated the ethoxyformic derivative to 100% of the value of the enzyme treated with only 40mM NH2OH. Since NH2OH specifically deacylates Nethoxyformylimidazole, it was shown that at least some of the histidine residues were essential for the activity. In addition, photooxidation experiments in the presence of 0.001% rose bengal confirmed that only histidine residues of the enzyme were destroyed under the designated experimental conditions. About two histidine residues per 37, 000g of protein were destroyed during the photooxidation experiments. In the presence of the competitive inhibitor carbobenzoxy-L-phenylalanine, ∗ only one histidine residue was destroyed, which inhibitor, that one histidine residue per 37, 000g of protein was essential for the enzyme activity.
