Lawson S L, Wakarchuk W W, Withers S G
Protein Engineering Network of Centres of Excellence, Department of Chemistry, University of British Columbia, Vancouver, Canada.
Biochemistry. 1997 Feb 25;36(8):2257-65. doi: 10.1021/bi9620215.
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.
糖苷酶所采用的作用机制部分取决于两个催化性羧酸之间的距离。在来自环状芽孢杆菌的保留型内切-β-1,4-木聚糖酶中，通过对推定的酸/碱催化剂Glu172进行诱变，改变了这个关键距离（约5.5埃）。距离增加（Glu172Asp）导致木聚糖水解的k(cat)值降低400倍。相比之下，通过对Glu172Cys突变体进行选择性羧甲基化实现距离减小，仅使木聚糖水解速率降低25倍。改变酸/碱催化剂的长度对芳基木二糖苷水解的不利影响较小，相对于野生型酶，k(cat)/Km值仅降低3至23倍。羧基的完全去除产生了更显著的影响。Glu172Cys和Glu172Gln突变体对需要酸催化的木聚糖或苯基木二糖苷底物没有可测量的活性。然而，这些突变体能够水解具有相对较好离去基团（pKa < 5.5）的芳基木二糖苷，这些底物几乎不需要质子辅助。叠氮化钠的添加导致Glu172Cys和Glu172Gln对2,5-二硝基苯基β-木二糖苷（pKa = 5.15）水解的速率显著增加。因此，通过添加阴离子亲核试剂可以部分弥补酸/碱催化剂的缺失。这些结果与Glu172在环状芽孢杆菌木聚糖酶中作为酸/碱催化剂发挥作用一致，并强调了该位置羧基的功能重要性。
