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Specificity of human cathepsin G
J Polanowska1, I Krokoszynska, H Czapinska
1Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland.
Biochimica Et Biophysica Acta
|July 24, 1998
Summary
Human cathepsin G shows dual trypsin- and chymotrypsin-like specificity, preferring specific amino acids at its S1 pocket. Subsite analysis revealed key mutations affecting inhibitor binding, offering insights into enzyme-inhibitor interactions.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Human cathepsin G is a serine protease implicated in various physiological and pathological processes.
- Understanding its substrate specificity is crucial for developing targeted inhibitors.
Purpose of the Study:
- To map the S1 binding pocket of human cathepsin G using synthetic substrates.
- To characterize the subsite specificity of cathepsin G using modified protein inhibitors.
Main Methods:
- Synthesis and kinetic analysis of tetrapeptide p-nitroanilide substrates.
- Site-directed mutagenesis of Cucurbita maxima trypsin inhibitor I (CMTI I).
- Determination of association constants for enzyme-inhibitor complex formation.
Main Results:
- Cathepsin G exhibits dual specificity, with a preference order of Lys=Phe>Arg=Leu>Met>Nle=Nva>Ala>Asp at the P1 position.
- Beta-branched side chains at P1 were deleterious.
- Mutations in CMTI I revealed specific residue requirements at P2, P3', and P1' for cathepsin G binding.
- Met8-->Arg substitution at P3' decreased cathepsin G binding, while Ala18-->Gly (P12') and Pro4-->Thr (P2) mutations increased it.
Conclusions:
- The S1 pocket of cathepsin G accommodates both basic and aromatic residues, indicating dual specificity.
- Subsite analysis provides a detailed map of interactions critical for cathepsin G inhibition.
- These findings are valuable for the rational design of selective cathepsin G inhibitors.