Fig. 6.

Proposed reaction mechanism for Pn3Pase and its E196A mutant (adapted from CAZypedia’s reaction mechanism for a retaining β-glycoside hydrolase). Pn3Pase operates with a double displacement mechanism which involves a glucuronyl-enzyme intermediate. The second step is the nucleophile attack of the glucuronyl-enzyme intermediate by a water residue, which is normally activated by E196. In the E196A mutant the water is likely activated, albeit less efficiently, by the carboxylate of the GlcA accounting for the small retention of activity when the acid//base catalytic residue is mutated.