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 succinogenes1,3-1,4-
![](/images/gifchars/beta2.gif)
-
D-glucanase (Fs
![](/images/gifchars/beta2.gif)
-glucanase), were characterized using site-directed mutagenesis, initial ratekinetics, fluorescence spectrometry,
and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in
Km value for lichenan was observed for W141F, W141H,
and W203R mutantFs
![](/images/gifchars/beta2.gif)
-glucanases,
and approximately 72-, 56-, 30-, 29.5-, 4.9-,
and 4.3-fold decreases in
kcat relative to thatfor the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F,
and W148Fmutants, respectively. In contrast, W186F
and W203F, unlike the other 12 mutants, exhibited a 1.4-
and4.2-fold increase in
kcat, respectively. W165F
and W203R were the only two mutants that exhibited a4-7-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 residuesretained a folding similar to that of the wild-type enzyme. Structural modeling
and kinetic studies suggestthat 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.