@article { ISI:A1994NN47200041,
	title = {MECHANISMS OF CELLULASES AND XYLANASES - A DETAILED KINETIC-STUDY OF THE EXO-BETA-1,4-GLYCANASE FROM CELLULOMONAS-FIMI},
	journal = {BIOCHEMISTRY},
	volume = {33},
	number = {20},
	year = {1994},
	month = {MAY 24},
	pages = {6363-6370},
	abstract = {The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates. This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism. Values of k(cat) are found to be invariant with pH whereas k(cat)/K-m is dependent upon two ionizations of pK(a) = 4.1 and 7.7. The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types. A strong dependence of both k(cat)(beta(1g) = -1) and k(cat)/K-m (beta(1g) = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting. For the cellobiosides a biphasic, concave downward plot is seen for k(cat) indicating a change in rate-determining step across the series. Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k(2) and log (k(2)/K-d) for the cellobiosides of modest (beta(1g) = -0.3) slope. These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states. Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta(1g) values, reveal that the presence of the distal. sugar moiety in cellobiosides results in a less highly charged transition state. These studies suggest that the primary function of the distal sugar is to increase the rate of formation of the glycosyl-enzyme intermediate through improved acid catalysis and greater nucleophile preassociation, without affecting its rate of decomposition.},
	issn = {0006-2960},
	doi = {10.1021/bi00186a041},
	author = {TULL, D and Withers, SG}
}
