PDBsum entry 1cb2

Hydrolase (o-glycosyl)

PDB id: 1cb2

Contents

Protein chains

363 a.a. *

Ligands

NAG ×4

MAN ×14

Waters ×392

* Residue conservation analysis

PDB id:

1cb2

Name:

Hydrolase (o-glycosyl)

Title:

Cellobiohydrolase ii, catalytic domain, mutant y169f

Structure:

Cellobiohydrolase ii. Chain: a, b. Fragment: catalytic. Synonym: cbh ii (y169f). Engineered: yes. Mutation: yes

Source:

Hypocrea jecorina. Organism_taxid: 51453. Gene: cbh2 (y169f). Expressed in: hypocrea jecorina. Expression_system_taxid: 51453.

Resolution:

2.00Å R-factor: 0.210 R-free: 0.232

Authors:

G.J.Kleywegt,M.Szardenings,T.A.Jones

Key ref:

A.Koivula et al. (1996). The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169. Protein Eng, 9, 691-699. PubMed id: 8875646

Date:

25-Nov-95 Release date: 14-Oct-96

Protein chains

P07987  (GUX2_HYPJE) -  Exoglucanase 2 from Hypocrea jecorina

Seq: 471 a.a.

Struc: 363 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.2.1.91 - cellulose 1,4-beta-cellobiosidase (non-reducing end).
• Reaction: Hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose and cellotetraose, releasing cellobiose from the non-reducing ends of the chains.

Protein Eng 9:691-699 (1996)

PubMed id: 8875646

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169.

A.Koivula, T.Reinikainen, L.Ruohonen, A.Valkeajärvi, M.Claeyssens, O.Teleman, G.J.Kleywegt, M.Szardenings, J.Rouvinen, T.A.Jones, T.T.Teeri.

ABSTRACT

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behavior of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 A resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.