Substrate specificity of endoglucanase A from Cellulomonas fimi: fundamental differences between endoglucanases and exoglucanases from family 6.
Values of kcat. and Km for the hydrolysis of cellotetraose, cellotriose, beta-cellobiosyl fluoride and various beta-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas fimi indicate that specific binding interactions between the reducing-end glucose residues of cellotetraose and cellotriose and the enzyme at the transition state provide enormous stabilization, endowing glucose with the "effective leaving group ability' of 2,4-dinitrophenol. As has been seen with several other inverting glycosidases, CenA hydrolyses the "wrong' anomer of its glycosyl fluoride substrate, alpha-cellobiosyl fluoride, according to non-Michaelian kinetics. This indicates that CenA carries out this hydrolysis by a mechanism involving binding of two substrate molecules in the active site (Hehre, Brewer and Genghof (1979) J. Biol. Chem. 254, 5942-5950] in contrast with that reported for cellobiohydrolase II, another family-6 enzyme [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 833-838]. The pH profiles for wild-type CenA indicate that kcat. for CenA depends on the presence of both a protonated group and a deprotonated group for full activity, consistent with the presence of an acid and a base catalyst at the active site. By contrast, the profile for the Asp252Ala mutant of CenA shows a dependence only on a base-catalytic group, thereby confirming the role of Asp-252 as an acid catalyst. These results show that hydrolysis by CenA occurs by a typical inverting mechanism involving both acid and base catalysis, as first proposed by Koshland. It also suggests that endoglucanases from family 6 may function by fundamentally different mechanisms for exoglucanases in this family.
Damude
H G
HG
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, Canada.
Ferro
V
V
Withers
S G
SG
Warren
R A
RA
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002475:Cellobiose / Q000737:chemistry; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry; D043326:Glucan 1,3-beta-Glucosidase; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013780:Tetroses / Q000737:chemistry; D014312:Trisaccharides / Q000737:chemistry; D001617:beta-Glucosidase / Q000378:metabolism*
D013780:Tetroses; D014312:Trisaccharides; D002475:Cellobiose; C048468:cellotetraose; C023872:mannotriose; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D043326:Glucan 1,3-beta-Glucosidase