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The roles of the residues in the catalytic trio Glu212-Asp214-Glu217 in
cellobiohydrolase I (CBHI) from Trichoderma reesei have been investigated by
changing these residues to their isosteric amide counterparts. Three mutants,
E212Q, D214N and E217Q, were constructed and expressed in T. reesei. All three
point mutations significantly impair the catalytic activity of the enzyme,
although all retain some residual activity. On the small chromophoric substrate
CNP-Lac, the kcat values were reduced to 1/2000, 1/85 and 1/370 of the wild-type
activity, respectively, whereas the KM values remained essentially unchanged. On
insoluble crystalline cellulose, BMCC, no significant activity was detected for
the E212Q and E217Q mutants, whereas the D214N mutant retained residual
activity. The consequences of the individual mutations on the active-site
structure were assessed for two of the mutants, E212Q and D214N, by X-ray
crystallography at 2.0 A and 2.2 A resolution, respectively. In addition, the
structure of E212Q CBHI in complex with the natural product, cellobiose, was
determined at 2.0 A resolution. The active-site structure of each mutant is very
similar to that of the wild-type enzyme. In the absence of ligand, the active
site of the D214N mutant contains a calcium ion firmly bound to Glu212, whereas
that of E212Q does not. This supports our hypothesis that Glu212 is the charged
species during catalysis. As in the complex of wild-type CBHI with bound
o-iodobenzyl-1-thio-beta-D-glucoside, cellobiose is bound to the two product
sites in the complex with E212Q. However, the binding of cellobiose differs from
that of the glucoside in that the cellobiose is shifted away from the trio of
catalytic residues to interact more intimately with a loop that is part of the
outer wall of the active site.
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