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Mutational Analysis of Thermus caldophilus GK24 ${\beta}$-Glycosidase: Role of His119 in Substrate Binding and Enzyme Activity  

Oh, Eun-Joo (Department of Genetic Engineering, Sungkyunkwan University)
Lee, Yoon-Jin (Department of Genetic Engineering, Sungkyunkwan University)
Choi, Jeong-Jin (Department of Genetic Engineering, Sungkyunkwan University)
Seo, Moo-Seok (Department of Genetic Engineering, Sungkyunkwan University)
Lee, Mi-Sun (Department of Genetic Engineering, Sungkyunkwan University)
Kim, Gun-A (Department of Genetic Engineering, Sungkyunkwan University)
Kwon, Suk-Tae (Department of Genetic Engineering, Sungkyunkwan University)
Publication Information
Journal of Microbiology and Biotechnology / v.18, no.2, 2008 , pp. 287-294 More about this Journal
Abstract
Three amino acid residues (His119, Glu164, and Glu338) in the active site of Thermus caldophilus GK24 ${\beta}$-glycosidase (Tca ${\beta}$-glycosidase), a family 1 glycosyl hydrolase, were mutated by site-directed mutagenesis. To verify the key catalytic residues, Glu164 and Glu338 were changed to Gly and Gln, respectively. The E164G mutation resulted in drastic reductions of both ${\beta}$-galactosidase and ${\beta}$-glucosidase activities, and the E338Q mutation caused complete loss of activity, confirming that the two residues are essential for the reaction process of glycosidic linkage hydrolysis. To investigate the role of His119 in substrate binding and enzyme activity, the residue was substituted with Gly. The H119G mutant showed 53-fold reduced activity on 5mM p-nitrophenyl ${\beta}$-D-galactopyranoside, when compared with the wild type; however, both the wild-type and mutant enzymes showed similar activity on 5mM p-nitrophenyl ${\beta}$-D-glucopyranoside at $75^{\circ}C$. Kinetic analysis with p-nitrophenyl ${\beta}$-D-galactopyranoside revealed that the $k_{cat}$ value of the H119G mutant was 76.3-fold lower than that of the wild type, but the $K_m$ of the mutant was 15.3-fold higher than that of the wild type owing to the much lower affinity of the mutant. Thus, the catalytic efficiency $(k_{cat}/K_m)$ of the mutant decreased to 0.08% to that of the wild type. The $k_{cat}$ value of the H119G mutant for p-nitrophenyl ${\beta}$-D-glucopyranoside was 5.l-fold higher than that of the wild type, but the catalytic efficiency of the mutant was 2.5% of that of the wild type. The H119G mutation gave rise to changes in optima pH (from 5.5-6.5 to 5.5) and temperature (from $90^{\circ}C\;to\;80-85^{\circ}C$). This difference of temperature optima originated in the decrease of H119G's thermostability. These results indicate that His119 is a crucial residue in ${\beta}$-galactosidase and ${\beta}$-glucosidase activities and also influences the enzyme's substrate binding affinity and thermostability.
Keywords
${\beta}$-Glycosidase; family 1 glycosyl hydrolase; Thermus caldophilus GK24; Tca ${\beta}$-glycosidase; site-directed mutagenesis; sequence analysis;
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