The active site structure of the Zn-containing neutral protease from Bacillus subtilis var. amylosacchariticus (BANP) was predicted by computer-aided modeling on the basis of the three-dimensional structure of thermolysin (TLN). As expected from the high homology in amino acid sequence of the two enzymes, the overall folding of BANP was very similar to that of TLN. Glu144, Tyr158, and His228 of BANP were located near the active site Zn ion, to which three amino acid residues, His143, His147, and Glu167, were coordinated. This model is supported by the previous results that chemical modifications of Tyr158 and photooxidation of His228 of BANP markedly affect the proteolytic activity of the enzyme. Interestingly, BANP was found to be significantly less sensitive to metalloprotease inhibitors such as phosphoramidon and talopeptin. From a comparison of the enzyme-inhibitor complex models between BANP and thermolysin, it is suggested that replacement of Thr129 in TLN by Phe130 in BANP is related to difference in inhibitor sensitivity between BANP and TLN.

The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0 degree C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. 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 enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.

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