title: Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme.
abstract: The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.
authors.affiliation: Institute of BioAgricultural Sciences, Academia Sinica, Taiwan, ROC.
authors.forename: Hsueh-Ling
authors.initials: HL
authors.lastname: Cheng
authors.forename: Li-Chu
authors.initials: LC
authors.lastname: Tsai
authors.forename: Su-Shiang
authors.initials: SS
authors.lastname: Lin
authors.forename: Hanna S
authors.initials: HS
authors.lastname: Yuan
authors.forename: Ning-Sun
authors.initials: NS
authors.lastname: Yang
authors.forename: Shu-Hua
authors.initials: SH
authors.lastname: Lee
authors.forename: Lie-Fen
authors.initials: LF
authors.lastname: Shyur
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan
chemical_list: D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014364:Tryptophan; D006026:Glycoside Hydrolases; C028020:licheninase