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

Koivula, Anu, Ruohonen, L., Wohlfahrt, G., Reinekainen, T., Teeri, T.T., Piens, K., Claeyssens, Marc, Weber, M., Vasella, A., Becker, D., Sinnott, Michael L., Zou, J., Kleywegt, G.J., Szardenings, M., Stahlberg, J. and Jones, T.A. (2002) The active site of cellobiohydralase Ce16A from Trichorderma reesei: the roles of aspartic acids D221 and D175. Journal of the American Chemical Society, 124 (34). pp. 10015-10024. ISSN 0002-7863

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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.

Item Type: Article
Additional Information: UoA 18 (Chemistry) © 2002 American Chemical Society
Subjects: Q Science > QD Chemistry
Schools: School of Applied Sciences
School of Applied Sciences > Biomolecular Sciences Research Centre
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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
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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
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Depositing User: Briony Heyhoe
Date Deposited: 13 Jul 2007
Last Modified: 10 Sep 2010 09:03


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