Role of subsite +1 residues in pH dependence and catalytic activity of the glycoside hydrolase family 1 beta-glucosidase BGL1A from the basidiomycete Phanerochaete chrysosporium.
The basidiomycete Phanerochaete chrysosporium produces two glycoside hydrolase family 1 intracellular beta-glucosidases, BGL1A and BGL1B, during the course of cellulose degradation. In order to clarify the catalytic difference between two enzymes, in spite of their high similarity in amino acid sequences (65%), five amino acids around the catalytic site of BGL1A were individually mutated to those of BGL1B (V173C, M177L, D229N, H231D, and K253A), and the effects of the mutations on cellobiose hydrolysis were evaluated. When the kinetic parameters (K(m) and k(cat)) were compared at the optimum pH for the wild-type enzyme, the kinetic efficiency was decreased in the cases of D229N, H231D, and K253A, but not V173C or M177L. The pH dependence of cellobiose hydrolysis showed a significantly more acidic pH profile for the D229N mutant, compared with the wild-type enzyme. Since D229 is located between K253 and the putative acid/base catalyst E170, we prepared the double mutant D229N/K253A, and found that its hydrolytic activity at neutral pH was restored to that of the wild-type enzyme. Our results indicate that the interaction between D229 and K253 is critical for the pH dependence and catalytic activity of BGL1A. Biotechnol. Bioeng.
Tsukada
Takeshi
T
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan.
Igarashi
Kiyohiko
K
Fushinobu
Shinya
S
Samejima
Masahiro
M
D019943:Amino Acid Substitution; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D020075:Phanerochaete / Q000201:enzymology*; D015202:Protein Engineering / Q000379:methods; D013329:Structure-Activity Relationship; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001617:beta-Glucosidase