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GH44 glycoside hydrolases are active on many substances, including tetrasaccharide cellooligosaccharides and longer oligomers, carboxymethylcellulose, xylan, lichenan, Avicel (slightly), and xyloglucan, the last of which appears to be a prime substrate [3, 4].
Kinetics and Mechanism
The most complete analyses of GH44 kinetics on various substrates are by Najmudin et al. [3] and by Warner et al. [4, 5]. GH44 endoglucanases are also xyloglucanases. They hydrolyze longer cellooligosaccharides faster than shorter cellooligosaccharides [4, 5]. They act asymmetrically on cellooligosaccharides, for instance producing more cellobiose and cellotetraose than cellotriose from cellohexaose [4, 5], with substrates bound with more of their residues in negatively-numbered than in positively-numbered subsites. Furthermore, disproportionation occurs, with more cellotetraose than cellobiose formed from cellohexaose, evidently caused by formation of larger unobserved products that are then rapidly hydrolyzed [4, 5]. GH44 enzymes act with retention of anomeric stereochemistry [1], through a classical Koshland double-displacement mechanism with a covalent bond being formed between the catalytic nucleophile and the anomeric carbon of the substrate, leading to liberation of the leaving group; subsequently, the glycosyl-enzyme is cleaved by water. A general acid/base residue acts as a general acid in the first step to assist departure of the aglycon; in the second step this residue acts as a general base to assist in deprotonating a nucleophilic water residue.
Catalytic Residues
The catalytic residues in this family have been suggested by several experiments with diverse enzymes. These include:
Clostridium thermocellum endoglucanase:General acid/base, Glu186; catalytic nucleophile, Glu359; by soaking the wild-type crystals with cellopentaose or cellohexaose and noting the positions of the residues relative to the reducing end of the cellotetraose product [1], and also by finding no activity with E186Q and E359Q mutants.
Protein from metagenomic library:General acid/base, Glu221; catalytic nucleophile, Glu393 by location in the active site of the wild-type crystal structure [2].
Clostridium acetobutylicum xyloglucanase/endoglucanase:General acid/base, Glu180; catalytic nucleophile, Glu352 also by location in the crystal structure of the wild-type enzyme, and by comparison with the C. thermocellum structure [5].
Three-dimensional structures
The first three-dimensional structure was by Kitago et al., who found a TIM-like barrel domain and a β-sandwich domain in C. thermocellum endoglucanase [1]. Similar structures were found by Nam et al. [2] in a protein from a metagenomic library and by Warner et al. [5] in C. acetobutylicum endoglucanase. Ca++ and Zn++ ions are found as ligands [1].
Kitago et al. [1] found that C. thermocellum endoglucanase acts by a retaining mechanism. They observed that a β-anomer was preferentially formed during cyclohexaitol hydrolysis.
Kitago et al. [1], by testing activity of the C. thermocellum endoglucanase E186Q mutant.
First 3-D structure
Kitago et al. [1] of C. thermocellum endoglucanase. It had a resolution of 0.96 Å and allowed the identification of the catalytic residues and the mechanism.
