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.
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.
Cheng
Hsueh-Ling
HL
Institute of BioAgricultural Sciences, Academia Sinica, Taiwan, ROC.
Tsai
Li-Chu
LC
Lin
Su-Shiang
SS
Yuan
Hanna S
HS
Yang
Ning-Sun
NS
Lee
Shu-Hua
SH
Shyur
Lie-Fen
LF
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
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014364:Tryptophan; D006026:Glycoside Hydrolases; C028020:licheninase