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Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2' of substrates.
- Source :
-
The FEBS journal [FEBS J] 2010 May; Vol. 277 (10), pp. 2255-67. - Publication Year :
- 2010
-
Abstract
- Chymases are chymotrypsin-like serine proteases that are found in large amounts in mast cell granules. So far, the extended cleavage specificities of eight such chymases have been determined, and four of these were shown to have a strong preference for acidic amino acids at position P2'. These enzymes have basic amino acids in positions 143 and 192 (Arg and Lys, respectively). We therefore hypothesized that Arg143 and Lys192 of human chymase mediate the preference for acidic amino acids at position P2' of substrates. In order to address this question, we performed site-directed mutagenesis of these two positions in human chymase. Analysis of the extended cleavage specificities of two single mutants (Arg143-->Gln and Lys192-->Met) and the combined double mutant revealed an altered specificity for P2' amino acids, whereas all other positions were essentially unaffected. A weakened preference for acidic amino acids at position P2' was observed for the two single mutants, whereas the double mutant lacked this preference. Therefore, we conclude that positions 143 and 192 in human chymase contribute to the strong preference for negatively charged amino acids at position P2'. This is the first time that a similar combined effect has been shown to influence the cleavage specificity, apart from position P1, among the chymases. Furthermore, the conservation of the preference for acidic P2' amino acids for several mast cell chymases clearly indicates that other substrates than angiotensin I may be major in vivo targets for these enzymes.
- Subjects :
- Amino Acid Sequence genetics
Amino Acid Substitution genetics
Animals
Chymases chemistry
Chymases genetics
Consensus Sequence genetics
Enzyme Activation
Humans
Mutagenesis, Site-Directed
Opossums
Peptide Library
Peptides chemistry
Peptides genetics
Peptides metabolism
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Substrate Specificity genetics
Thioredoxins genetics
Amino Acids, Acidic chemistry
Arginine chemistry
Chymases metabolism
Lysine chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1742-4658
- Volume :
- 277
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- The FEBS journal
- Publication Type :
- Academic Journal
- Accession number :
- 20423454
- Full Text :
- https://doi.org/10.1111/j.1742-4658.2010.07642.x