Removal of chondroitin sulfate glycosaminoglycan (GAG) chains with chondroitinase ABC I (chABC I) in CNS injury models promotes both saxon regeneration and plasticity. It has been suggested that direct interaction between an aromatic pair appears to contribute about - 1.3 kcal/mol to the stability of a folded protein, so introducing an aromatic pair by point mutation might increase the enzyme activity and thermal stability as in the case of mesophilic xylanase, although using this approach destabilized T4 lysozyme. In this study, we used site-directed mutagenesis to investigate the effect of new aromatic pairs on activity and stability of chABC I. We replaced Ile295, Ser581, and Gly730 adjacent to pre-existing aromatic residues with Tyr to obtain new aromatic pairs, i.e., Tyr295/His372, Tyr576/Tyr581, and Tyr623/Tyr730. Results showed that Km values of S581Y and G730Y variants decreased relative to wild-type enzyme while their catalytic efficiency (kcat/Km) increased but I295Y variant was inactive. Also, long-term and thermal stability of the active mutants was decreased. Fluorescence and circular dichroism studies showed that these mutations resulted in a more flexible enzyme structures: a finding which was confirmed by thermal and limited proteolytic studies. In conclusion, the activity of chABC I can be improved by introducing appropriate aromatic pairs at the enzyme surface. This approach did not provide any promising results regarding the enzyme stability.
Submitter: Shu-Ching Ou
Submission Date: March 8, 2019, 12:50 p.m.
|Number of data points||49|
|Proteins||Chondroitin sulfate ABC endolyase|
|Assays/Quantities/Protocols||Experimental Assay: Km ; Experimental Assay: Vmax ; Experimental Assay: kcat ; Experimental Assay: kcat/Km ; Experimental Assay: Secondary Structure: α-helix percentage ; Experimental Assay: Secondary Structure: β-structure percentage ; Experimental Assay: Secondary Structure: β-turn percentage ; Experimental Assay: Secondary Structure: Random coil percentage ; Experimental Assay: Stern–Volmer constant KSV (acrylamide) ; Experimental Assay: Stern–Volmer constant KSV (KI)|
|Libraries||Variants for chABC-I_C4S ; Variants for chABC-I|