Tsukada Takeshi, Igarashi Kiyohiko, Fushinobu Shinya, Samejima Masahiro
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan.
Biotechnol Bioeng. 2008 Apr 15;99(6):1295-302. doi: 10.1002/bit.21717.
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.
担子菌黄孢原毛平革菌在纤维素降解过程中产生两种糖苷水解酶家族1的细胞内β-葡萄糖苷酶，即BGL1A和BGL1B。为了阐明这两种酶之间的催化差异，尽管它们在氨基酸序列上具有高度相似性（65%），但仍将BGL1A催化位点周围的五个氨基酸分别突变为BGL1B的相应氨基酸（V173C、M177L、D229N、H231D和K253A），并评估了这些突变对纤维二糖水解的影响。在野生型酶的最适pH下比较动力学参数（K(m)和k(cat)）时，D229N、H231D和K253A突变体的动力学效率降低，但V173C或M177L突变体未出现这种情况。与野生型酶相比，D229N突变体的纤维二糖水解pH依赖性显示出明显更偏酸性的pH曲线。由于D229位于K253和推定的酸碱催化剂E170之间，我们制备了双突变体D229N/K253A，发现其在中性pH下的水解活性恢复到了野生型酶的水平。我们的结果表明，D229和K253之间的相互作用对于BGL1A的pH依赖性和催化活性至关重要。《生物技术与生物工程》
