The active site of cellobiohydralase Ce16A from Trichorderma reesei: the roles of aspartic acids D221 and D175

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have
established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that
are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other.
Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to
confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to
contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural
and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve
a catalytic base. The value of D2O(V) of 1.16 ( 0.14 for hydrolysis of cellotriose suggests that the large
direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus
mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond
between D221 and D175 before catalysis.