title: Chemical modification of neutral protease from Bacillus subtilis var. amylosacchariticus with tetranitromethane: assignment of tyrosyl residues nitrated.
abstract: A neutral protease from Bacillus subtilis var. amylosacchariticus was modified with tetranitromethane (TNM) at pH 8.0 for 1 h at 25 degrees C, by which treatment the proteolytic activity toward casein was markedly reduced, whereas activity changes toward N-blocked peptide substrates were variable depending upon the substrate used. The modified enzyme was digested with a Staphylococcus aureus V8 protease at pH 7.9 and the resultant peptides were separated by HPLC. Two peptides which contain nitrotyrosyl residue(s) were purified. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153-159 of the neutral protease, and Tyr-158 was identified as PTH-nitrotyrosine. The other one was the amino-terminal peptide of residue Nos. 1-22, and Tyr-21 was shown to be nitrated. From a comparison with the active site structure of thermolysin, which is a zinc metalloprotease with a high sequence homology to B. subtilis neutral proteases, nitration of Tyr-158 was inferred to be closely related to the activity changes of the neutral protease from B. subtilis var. amylosacchariticus.
authors.affiliation: School of Pharmaceutical Sciences, Nagasaki University.
authors.forename: R
authors.initials: R
authors.lastname: Kobayashi
authors.forename: A
authors.initials: A
authors.lastname: Kanatani
authors.forename: T
authors.initials: T
authors.lastname: Yoshimoto
authors.forename: D
authors.initials: D
authors.lastname: Tsuru
mesh_terms: D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000378:metabolism*; D008697:Methane / Q000031:analogs & derivatives*; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism; D013774:Tetranitromethane / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D014443:Tyrosine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D013774:Tetranitromethane; D008697:Methane