Institute of BioAgricultural Sciences, Academia Sinica, Taiwan, R.O.C., Department of Nursing, Mei Ho Institute of Technology, Taiwan, R.O.C., and Institute of Molecular Biology, Academia Sinica, Taiwan, R.O.C.

Abstract:  

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp⁵⁴, Trp¹⁰⁵, Trp¹¹², Trp¹⁴¹, Trp¹⁴⁸, Trp¹⁶⁵, Trp¹⁸⁶, Trp¹⁹⁸, and Trp²⁰³ in Fibrobacter succinogenes 1,3-1,4--D-glucanase (Fs-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-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 Trp⁵⁴, Trp¹⁴¹, Trp¹⁴⁸, and Trp²⁰³ play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.