Inactivation of Streptomyces subtilisin inhibitory by chemical modifications.
1. The inhibitory activity of an alkaline protease inhibitor, (Streptomyces subtilisin inhibitor) towards subtilisin is found to decrease by photooxidation sensitized by methylene blue with a clear pH dependence, the midpoint of which is about 6.0. 2. Amino acid analyses of photooxidized Streptomyces subtilisin inhibitor indicate that one of the two histidyl residues and the three methionyl residues are destroyed, concomittant with the loss of inhibitory activity. 3. In accordance with this observation, one of the clearly resolved nuclear magnetic resonances from C2-protons of the two histidyl residues is selectively diminished. This histidyl residue, sensitive to photooxidation and giving a proton magnetic resonance peak at lower field, is assigned to His-106 from peptide analyses. 4. Independent modification of methionyl residues by a reaction with H2O2 or Cl2 also decreases the inhibitory activity of Streptomyces subtilisin inhibitor. 5. Modification of lysyl, tyrosyl and tryptophanyl residues by diazonium-1-H-tetrazole does not lead to the loss of the inhibitory activity. 6. The above results indicate that one or more methionyl residue(s) are essential to the inhibitory activity of Streptomyces subtilisin inhibitor, whereas lysyl, tyrosyl and tryptophanyl residues are not essential to the inhibitory activity. Modification of His-106 is also strongly related to the loss of activity, although its distinct participation in the inactivation mechanism has not been demonstrated.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000494:pharmacology; D002713:Chlorine; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013302:Streptomyces / Q000032:analysis*; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D001426:Bacterial Proteins; D004220:Disulfides; D010446:Peptide Fragments; D002713:Chlorine; D006861:Hydrogen Peroxide; D013381:Subtilisins; D003488:Cyanogen Bromide