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

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Table 1. Kinetic Parameters for the wt Cel6A and Mutant Enzymes D221A and D175A on Cello-oligosaccharides (Glcn, n ) 4-6)
Measured in 10 mM Sodium Acetate Buffer, pH 5.0 at 27 °Ca
wt Cel6A D221A D175A
substrate kcat (s-1) Km (íM) kcat/Km (s-1 íM-1) kcat (s-1) kcat (s-1) Km (íM) kcat/Km (s-1 íM-1)
Glc3 0.06 ( 0.01 17 ( 5 0.004 ( 0.002 e0.0001 e0.0003 nd
Glc4 4.1 ( 0.5 2.6 ( 0.5 2.0 ( 0.5 e0.0012 0.0013 ( 0.0010 4 ( 1 0.0003 ( 0.0004
Glc5 1.1 ( 0.2 1.3 ( 0.4 0.8 ( 0.5 e0.0010 0.025 ( 0.001 1.4 ( 0.7 0.02 ( 0.01
Glc6 14 (2 14 ( 6 1.0 ( 0.7 e0.0007 0.020 ( 0.001 nd
aOwing to low activity, Km values could not be measured for the D221A mutant. Kinetic constants were calculated by a nonlinear regression data analysis
program (Enzfit), which also gives the standard deviation. nd ) not determined.
Catalytic Mechanism of T. reesei Cel6A A R T I C L E S
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Figure 5. Snapshots of the molecular dynamics simulations of the proposed catalytic states. Only the residues in the -1 and +1 sites are shown. All
hydrogens and the backbone atoms of Y169, D175, and D221 are omitted for clarity. Important hydrogen bonds are shown as green dots, and the interaction
between water and C1, important for the nucleophilic attack, is shown in orange. The models of different reaction states show (a) the ground state with the
-1 site sugar in a skew-boat conformation (2S0), (b) the ground state with the D221 side chain rotated toward the glycosidic oxygen, (c) an oxycarbenium
ion intermediate modeled in a boat conformation (2,5B) as an analogue to the transition state, and (d) two sugars of the reaction products (R-cellobiose in the
-1 and -2 sites and â-cellobiose in the +1 and +2 sites).
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