Send to

Choose Destination
See comment in PubMed Commons below
Biochemistry. 1997 Feb 25;36(8):2257-65.

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

Author information

  • 1Protein Engineering Network of Centres of Excellence, Department of Chemistry, University of British Columbia, Vancouver, Canada.


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 (approximately 5.5 A) 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 Glu172Cys 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)/Km 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 (pKa < 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 (pKa = 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.

[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society
    Loading ...
    Write to the Help Desk