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Positioning the acid/base catalyst in a glycosidase: Studies with Bacillus circulans xylanase

TitlePositioning the acid/base catalyst in a glycosidase: Studies with Bacillus circulans xylanase
Publication TypeJournal Article
Year of Publication1997
AuthorsLawson, SL, WAKARCHUK, WW, Withers, SG
JournalBIOCHEMISTRY
Volume36
Pagination2257-2265
Date PublishedFEB 25
ISSN0006-2960
Abstract

The mechanism of action employed by a glycosidase is dictated, in part, by the distance between the two catalytic carboxylic acids, In the retaining endo-beta-1,4-xylanase from Bacillus circulans, this critical distance (similar to 5.5 Angstrom) has been altered by mutagenesis of the putative acid/base catalyst Glu172. An increase in the separation (Glu172Asp) resulted in a 400-fold decrease in the k(cat) value for xylan hydrolysis. By contrast, a decrease in the separation, achieved by the selective carboxymethylation of the Glu 172Cys mutant, caused only a 25-fold reduction in the rate of xylan hydrolysis, Altering the length of the acid/base catalyst had a less detrimental effect on the hydrolysis of aryl xylobiosides, with k(cat)/K-m values being reduced only 3-23-fold relative to the wild-type enzyme. Complete removal of the carboxyl group had a more dramatic effect. The Glu172Cys and Glu172Gln mutants exhibited no measurable activity on xylan or phenyl xylobioside, substrates which require acid catalysis. However, these mutants were capable of hydrolyzing aryl xylobiosides with relatively good leaving groups (pK(a) <5.5), which need Little protonic assistance. The addition of sodium azide caused significant rate increases for the hydrolysis of 2,5-dinitrophenyl beta-xylobioside (pK(a)=5.15) by Glu172Cys and Glu172Gln. Thus, the absence of an acid/base catalyst can be partially compensated for by the addition of an anionic nucleophile. These results are consistent with Glu172 functioning as the acid/base catalyst in B. circulans xylanase and emphasize the functional importance of the carboxyl group found at this position.

DOI10.1021/bi9620215