Positioning the Acid/Base Catalyst in a Glycosidase:  Studies with Bacillus circulans Xylanase

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 (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.

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

“…Another conserved glutamic acid residue identified in a conserved motif of [G-(T/F)-E*] was also recognized as acid-base residue catalysts in the BLAST search comparing twelve known GH-12 cellulase sequences performed [73]. Comparisons with other retaining glycosidases revealed that a general acid-base catalytic mechanism is less consistent compared with a nucleophile catalytic mechanism [75,76]. Both the nucleophile conserved sequence (E*-(L)-M-(I)-W) and the acid-base residues sequence (G-(T)-E*) have been detected in cloned celB (amino acid residues 178-182 and 244-246, respectively).…”

Measuring “Unmeasurable” Folding Kinetics of Proteins by Single-Molecule Force Spectroscopy

“…BCX has been the subject of much previous study using a variety of techniques including X-ray crystallography, NMR spectroscopy, and mutational analysis, leading to a well-defined structure and enzymatic mechanism, 12,13 including the identification of structural elements critical to cleavage of xylan substrates. [14][15][16][17][18] Despite the extensive characterization of BCX, previous attempts to characterize its folding kinetics using thermal denaturation failed due to aggregation upon unfolding; 4 attempts using chemical denaturation have encountered only limited success. 17,19 Because aggregation is highly dependent on protein concentration, refolding under dilute conditions has been used to alleviate such problems.…”

“…[14][15][16][17][18] Despite the extensive characterization of BCX, previous attempts to characterize its folding kinetics using thermal denaturation failed due to aggregation upon unfolding; 4 attempts using chemical denaturation have encountered only limited success. 17,19 Because aggregation is highly dependent on protein concentration, refolding under dilute conditions has been used to alleviate such problems. 4,20 In principle, if the concentration of unfolded proteins is sufficiently low, the rate of association between unfolded protein molecules (a second-order process) becomes slower than the rate of refolding (typically a first-order process), eliminating the possibility of aggregation.…”

A Secondary Xylan-binding Site Enhances the Catalytic Activity of a Single-domain Family 11 Glycoside Hydrolase

“…14,15 Bacillus circulans xylanase (BcX) is a ∼20 kDa retaining endo-1,4-β-xylanase of the glycoside hydrolase family 11, clan GH-C †. 16 The enzyme has been extensively characterized kinetically, [17][18][19][20][21] as well as structurally by X-ray crystallography and NMR spectroscopy. [22][23][24] The β-sandwich protein has a fold resembling that of a partially closed right hand, with the active site "palm" formed by the cleft between the "fingers" and "thumb".…”