Mutagenesis of Trp54 and Trp203 Residues on Fibrobacter Succinogenes 1,3-1,4--
详细信息 查看全文
- 作者：Hsueh-Ling Cheng ; Li-Chu Tsai ; Su-Shiang Lin ; Hanna S. Yuan ; Ning-Sun Yang ; Shu-Hua Lee ; and Lie-Fen Shyur
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：July 9, 2002
- 年：2002
- 卷：41
- 期：27
- 页码：8759 - 8766
- 全文大小：182K
- 年卷期：v.41,no.27(July 9, 2002)
- ISSN：1520-4995
文摘
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 succinogenes1,3-1,4-
-D-glucanase (Fs-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-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 Trp54, Trp141, Trp148, and Trp203 play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.
-D-glucanase (Fs-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-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 Trp54, Trp141, Trp148, and Trp203 play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.