Rapid determination of substrate specificity of Clostridium histolyticum β-collagenase using an immobilized peptide library

Abstract

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 (kcat/Km) 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 (P3-P4′) 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 P4′, and an aromatic side chain is preferred in this position. The optimal P1′-P3′ extended substrate sequence is P1′-Gly/Ala, P2′-Pro/Xaa, and P3′-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P2 or P3 position is required for a high substrate activity with C. histolyticum β-collagenase. S2 and S3 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.