Mode of action of endoglucanase III from Trichoderma reesei.
Endoglucanase III (EG III) was purified to homogeneity from the culture medium of Trichoderma reesei QM 9414. It has a molecular mass of 48 kDa, and an isoelectric point of 5.1. Maximal activity was observed between pH4 and 5. Celloligosaccharides and their chromophoric derivatives were used as substrates, and the reaction products were analysed by quantitative h.p.l.c. Nucleophilic competition experiments (between methanol and water) allowed unequivocal assessment of cleavage sites. EG III preferentially released cellobiose (or the corresponding glycoside) from the reducing end of the higher cellodextrins. A putative binding model containing five subsites is proposed. The pH-dependence of 4'-methylumbelliferyl beta-cellotrioside hydrolysis indicates the presence of a protonated group with a pK 5.5 in the reaction mechanism, and the possible involvement of a carboxy group is corroborated by a temperature study (delta Hion = -15.9 J/mol). This, together with independent evidence from affinity-labelling experiments [Tomme, Macarrón and Claeyssens (1991) Cellulose '91, New Orleans, Abstr. 32] and n.m.r. studies [Gebbler, Gilkes, Claeyssens, Wilson, Béguin, Wakarchuk, Kilburn, Miller, Warren and Withers (1992) J. Biol. Chem. 267, 12559-12561], favours the assumption of a lysozyme-type (retention of configuration, two essential carboxy groups) mechanism for this family A cellulase.
Macarrón
R
R
Department of Biochemistry, Faculty of Chemistry, Complutense University, Madrid, Spain.
Acebal
C
C
Castillón
M P
MP
Domínguez
J M
JM
de la Mata
I
I
Pettersson
G
G
Tomme
P
P
Claeyssens
M
M
D001426:Bacterial Proteins; D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006923:Hymecromone / Q000031:analogs & derivatives; D007526:Isoelectric Point; D007700:Kinetics; D008954:Models, Biological; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
D001426:Bacterial Proteins; D005960:Glucosides; D009844:Oligosaccharides; D006923:Hymecromone; D002482:Cellulose; D002480:Cellulase; C061269:cel-3 protein, Fibrobacter succinogenes