Abstract
The amino acid residues responsible for the enzyme activity of semi-alkaline proteinase from Aspergillus melleus were identified by means of chemical modification studies. The modification of the enzyme with N-bromosuccinimide (NBS) resulted in the loss of the enzyme activity and a subtle alteration of conformation. NBS-modified enzyme still retained the antigenic structure, but became labile to heat and pH as the extent of modification of tryptophan increased. The relation between the extent of tryptophan oxidation and the enzyme stability suggested that 1 of the 3 tryptophan residues is important for the maintenance of structural integrity of the enzyme. The dye-sensitized photooxidation of the enzyme led to the loss of the enzyme activity with first-order kinetics. The rate of inactivation of this enzyme was pH-dependent and the rate constant-pH profile gave a sigmoidal curve with an inflection point at pH 6.5. Amino acid analysis of photooxidized enzyme indicated that the inactivation of this enzyme was directly proportional to the loss of histidine residue. Thus, these results suggested that at least 1 histidine residue is involved in the active site of the enzyme.
