Ketone-substrate analogues of Clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors

A series of ketone-substrate analogues has been synthesized for the two classes of collagenases from Clostridium histolyticum and shown to be competitive inhibitors. These compounds have sequences that match those of specific peptide substrates for these enzymes. The best inhibitor is the ketone analogue of cinnamoyl-Leu-Gly-Pro-Pro, which has a KI value of 18 nM for epsilon-collagenase, a class II enzyme. This is the tightest binding inhibitor reported for any collagenase to date. Plots of log KI for the inhibitors vs log KM/kcat for the matched substrates for both collagenases are linear with slopes near unity, indicating that the ketones are transition-state analogues. This strongly implies that the ketone carbon atoms of these inhibitors are tetrahedral when bound to the enzymes.