Substrate Recognition and Processing

The Gly493-494 Motif allows only glycine to adopt the P1' position, and prevents short peptides binding


The S1’ recognition site (Gly493-Gly494), allows only a glycine residue to adopt the P1’ position in the collagen molecule, which follows a Gly-Pro-X repeat motif. This site also forms a secondary S1’ oxyanion pocket as their amides point towards the P1’ carbonyl oxygen of the substrate. This forms a strong S1’-P1’ oxyanion interaction (figure 1.)


The secondary oxyanion pocket causes a strong S1'-P1' interaction, which misaligns the position of the zinc atom in the catalytic site unless P2 and P3 residues occupy the other recognition sites. Therefore enzymatic activity on substrates containing P2 and P3 residues is much more efficient and preferable.




The wide cleft in the substrate recognition site accommodates different amino acids


There is a degree of flexibility due to a wide cleft at the S3 to S1 substrate recognition sites allowing alternation between the exact residue identities of the P3 to P1 substrate amino acids such as making allowances for prolines and hydroxyprolines which cause a kinking in the collagen structure which cannot normally be accommodated

A wall is present to act as the 'molecular ruler'


A loop segment (Gln511-Phe515) functions at the entrance to the primed substrate recognition sites, precedes the central TLP-like fold helix, which serves as a molecular ruler due to making a kink at the P4’ position of longer substrates making binding energetically less favourable (keeps recognition down to 3 peptides at a time).

Figure.1 Shows us the active site of Collagenase G and the specific functional areas

· Two step mechanism:

1. CLOSED CONFORMATION: Activator domain interacts with the peptidase domain allowing processing of both triple helices and microfibrils. These domains are normally separated by 40Å ,which constitutes the open conformation. The closed conformation occurs when two major contacts form at the bottom of the saddle in response to collagen binding to the active site and an alternative four helix bundle arrangement occurs, allows the HEAT protein recognition motifs to interact with the triple helical collagen and unwind the chains which are then cleaved. Ground state of collagenase has a perfect match with the dimensions of collagen microfibrils which have a diameter of 40 angstroms. This allows a squeezing of the fibril on transition to the closed state like a pair of pliers, allows processing of single collagen triple helices. 
2.  RESETTING: After processing, the open state allows rest of microfibril to enter collagenase.