IDENTIFICATION OF ACTIVE-SITE CARBOXYLIC RESIDUES IN BACILLUS-LICHENIFORMIS 1,3-1,4-BETA-D-GLUCAN 4-GLUCANOHYDROLASE BY SITE-DIRECTED MUTAGENESIS

Active site residues of 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3 .2.1.73) from Bacillus licheniformis have been identified by site-dire cted mutagenesis. Previous work revealed that Glu-134 was essential fo r enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaci ens enzyme. To search for the general acid catalyst, the Asp and Glu r esidues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138 Q mutation yielded an inactive enzyme, whereas the E134Q and and D136N mutants retained less than 0.5% of the wild type activity Based on th e three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillu s macerans 1,3-1,4-beta-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly l ocated into the active site cleft to participate in catalysis. Glu-138 appears as the most Likely candidate to function as the general acid catalyst, while Asp-136 may affect the pK(a) of the catalytic residues .