• Applied Biological Chemistry
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• v.40 no.3
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• pp.189-195
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• 1997

The structural basis of Iysine specificity of Achromobacter protease I (API) was investigated by means of site-directed mutagenesis. The precursor protein in which Glu190, one of the two candidates for determining Iysine specificity, was substituted by glutamine, aspartic acid or leucine was processed autocatalytically to attaln full pretense activity with lysine specificity. The substitution of the other candidate, Asp225, for asparagine or leucine produced no mature active forms of pro-API. The precursor protein of the mutant D225E slowly matured autocatalytically. The lysylendopeptidase activity of the mature D225E was 0.25% of that of native API, and this reduced activity is mainly due to a decrease in the affinity of the enzyme for lysine. These results suggest that Asp225 plays a critical rol in restricted substrate specificity as a lysylendopeptidase. However, D225E exhibited no measurable activity for synthetic ornithine substrate. Since the hydroxyl group of Ser194 in this mutant retained essentially the same reactivity to DFP or PMSF as that in native API, it can be noted that a methylene unit longer side chain of residue 225 is not compensated by a methylene unit shorter side chain at subsite P1 in the bound substrate.

• Applied Biological Chemistry
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• v.40 no.3
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• pp.196-201
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• 1997

Assuming that Asp225 is the substrate specificity determinant of Achromobacter pretense I (APl) which is lysine-specific serine protease, the 225th residue was substituted for other amino acids with a hope that the substrate specificity of a mutant API is altered. Furthermore, to maturate preform of mutant API autocatalytically, Lys(-1) was also replaced by Met, Asp, or Glu. However, all the mutants were not expressed, or accumulated as inactive precursor proteins. This result implicats that Asp225 plays a critical rol in restricted substrate specificity as a lysylendopeptidase but the substrate specificity of API is not determined only by the nature of residue 225.