TY - JOUR
T1 - Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-β-D-glucan 4-glucanohydrolase by site-directed mutagenesis
AU - Juncosa, Miquel
AU - Pons, Jaume
AU - Dot, Teresa
AU - Querol, Enrique
AU - Planas, Antoni
PY - 1994/5/20
Y1 - 1994/5/20
N2 - Active site residues of 1,3-1,4-β-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3-1,4-β-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located 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(α) of the catalytic residues.
AB - Active site residues of 1,3-1,4-β-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3-1,4-β-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located 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(α) of the catalytic residues.
UR - http://www.scopus.com/inward/record.url?scp=0028225393&partnerID=8YFLogxK
M3 - Article
C2 - 8182059
AN - SCOPUS:0028225393
SN - 0021-9258
VL - 269
SP - 14530
EP - 14535
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 20
ER -