Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases.
The catalytic activity of nine enzymes (endoglucanases I-III, V, VI and cellobiohydrolases I and II from Humicola insolens; endoglucanases A and C from Bacillus lautus), representative of cellulase families A-C, H, J and K, has been investigated using a series of reduced cellooligosaccharides (cellotriitol to cellohexaitol) as substrates. For each enzyme, the specificity of cleavage was determined by analytical HPLC while the kinetic constants were obtained from a kinetic assay involving a cellobiose dehydrogenase purified from H. insolens as a coupled enzyme using 2,6-dichloroindophenol as the electron acceptor. These data were used to estimate the number of subsites in the enzymes. The stereochemical course of hydrolysis by seven enzymes, representing the six different families, was assessed using 1H-NMR. The enzymes belonging to families which had already been investigated (A-C), showed results in agreement with previous studies. The three other families (H, J and K), for which no mechanistic data was previously available, gave results which indicated that enzymes in group H had retaining-type activity and enzymes in groups J and K had inverting-type activity. The retaining endoglucanases I and III displayed a high glycosyl-transferase activity under the conditions used during the NMR experiments resulting in precipitates of higher oligomers.
Schou
C
C
Department of Biochemistry and Nutrition, Technical University of Denmark, Lyngby.
Rasmussen
G
G
Kaltoft
M B
MB
Henrissat
B
B
Schülein
M
M
D001407:Bacillus / Q000201:enzymology; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003912:Dextrins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D003904:Mitosporic Fungi / Q000201:enzymology; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D013379:Substrate Specificity
D003912:Dextrins; D002482:Cellulose; C068994:cellodextrin; D002480:Cellulase