Rapid Determination of Substrate Specificity of Clostridium histolyticum β-Collagenase Using an Immobilized Peptide Library

• Published in: The Journal of biological chemistry Vol. 277; no. 10; p. 8366
• Main Authors: Yongbo Hu, Erin Webb, Jasbir Singh, Barry A. Morgan, James A. Gainor, Thomas D. Gordon, Teruna J. Siahaan
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 08-03-2002
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111042200

The molecular basis of the substrate specificity of Clostridium histolyticum β-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000
sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each
sequence. This immobilized peptide library was incubated with C. histolyticum β-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity ( k
cat / K
m ) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing
was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites
(P 3 –P 4 ′) of C. histolyticum β-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the
activity of the enzyme is P 4 ′, and an aromatic side chain is preferred in this position. The optimal P 1 ′–P 3 ′ extended substrate sequence is P 1 ′-Gly/Ala, P 2 ′-Pro/Xaa, and P 3 ′-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P 2 or P 3 position is required for a high substrate activity with C. histolyticum β-collagenase. S 2 and S 3 sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to
be a powerful approach for assessing the substrate specificity of C. histolyticum β-collagenase, so it may be applied to the study of other proteases of interest.