SUBSTRATE-SPECIFICITY OF ENDOGLUCANASE-A FROM CELLULOMONAS-FIMI - FUNDAMENTAL DIFFERENCES BETWEEN ENDOGLUCANASES AND EXOGLUCANASES FROM FAMILY-6

Values of k(cat). and K-m for the hydrolysis of cellotetraose, cellotr iose, beta-cellobiosyl fluoride and various beta-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas funi indicate that specific binding interactions between the reducing-end glucose residues of cell otetraose and cellotriose and the enzyme at the transition state provi de enormous stabilization, endowing glucose with the 'effective leavin g group ability' of 2,4-dinitrophenol. As has been seen with several o ther 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 hy drolysis by a mechanism involving binding of two substrate molecules i n 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) Bi ochem. J. 291, 833-838]. The pH profiles for wild-type CenA indicate t hat k(cat.) for CenA depends on the presence of both a protonated grou p and a deprotonated group for full activity, consistent with the pres ence of an acid and a base catalyst at the active site. By contrast, t he 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 from exog lucanases in this family.