Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei

FEBS Letters

Claeyssens, Marc¹  Tomme, Peter¹ 

[1] Laboratorium voor Biochemie, Rijksuniversiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium

关键词: Cellulase;    Cellobiohydrolase;    Active site;    Modification;    Homology;    Labeling;    CBH I;    cellobiohydrolase (EC 3.2.1.91);    core I;    core-protein of CBH I;    EG;    endoglucanase (EC 3.2.1.4);    WRK;    N-ethyl-5-phenylisoxazolium-3′-sulfonate;    DEP;    diethylpyrocarbonate;    NBS;    N-bromosuccinimide;    TNM;    tetranitromethane;    Mes;    N-morpholinomethane-sulfonic acid;    Tris;    tris(hydroxymethyl)aminomethane;    CNPL;    2′-chloro-4′-nitrophenyl β-lactoside;    TFA;    trifluoroacetic acid;    RP-HPLC;    reversed-phase high pressure liquid chromatography;    DTT;    dithiothreitol;    GU.HCl;    guanidine hydrochloride;   

DOI  :  10.1016/0014-5793(89)80136-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Several aspects of the specific modification of cellobiohydrolase I (CBH I) from Trichoderma reesei with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3′-sulfonate) are presented. The pH dependence of the resulting inactivation points to the implication of an ionising group with a pK _a of approx. 5.5. The rapid inactivation kinetics, the specific protection and the stoichiometry of modification (3 versus 2 residues), together with the isolation and amino acid sequencing of the putative active site peptide, provide a large body of evidence for the presence of a catalytically important carboxyl residue in the 125–135 region of the CBH I amino acid sequence. From the striking homology between this peptide sequence and those of the active site regions of different lysozymes, glutamic acid 126 is retained as the most plausible catalytic residue (proton donor) in CBH I, equivalent to glutamic acid 35 in hen egg white lysozyme. Glutamic acid 127 is proposed as a potential active site residue to the homologous endoglucanase I (EG I) isolated from the same Trichoderma species.

【 授权许可】

Unknown