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研究生:池俊利
研究生(外文):Jiunly Chir
論文名稱:Flavobacteriummeningosepticum中第三家族β-葡萄糖甘酵素的反應機制研究及其親核性基團與一般酸鹼催化基團之鑑定
論文名稱(外文):Mechanistic study and direct evidence on identification of the nucleophile and acid/base catalyst of a family 3 β-glucosidase from Flavobacterium meningosepticum
指導教授:李耀坤李耀坤引用關係
指導教授(外文):李耀坤
學位類別:博士
校院名稱:國立交通大學
系所名稱:應用化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:90
語文別:中文
中文關鍵詞:β-葡萄糖甘酵素親核基一般酸鹼催化之胺基酸機制
外文關鍵詞:β-glucosidaseNucleophile residuegeneral acid/base catalyst residuemechanism
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本研究旨在探討對自黃質菌 (Flavobacterium meningosepticum) 中選殖之family 3 β-葡萄糖甘酵素進行性質與反應機制之研究 (第三章) 及其親核性基團 (第四章 )與一般酸鹼催化基團 (第五章) 之鑑定。
基因重組及所有突變酵素均可由E.coli 細胞粗提取液經硫酸銨鹽沉澱,再以HiTrap SP陽離子交換樹脂管柱於 pH 6.9 下進行管柱層析分離而得純度可達 90 % 以上之酵素,其單體分子量約為79 kDa。以野生株 (wild type) 酵素而言,在可測量的範圍內,反應最佳之酸鹼值為4.5~5.0。此酵素對受質的特異性為糖基部份的選擇性甚嚴,但對非糖基部份的選擇性則較寬鬆。葡萄糖 (產物之一) 對此酵素有抑制效果,Ki 為4.6 mM,而δ-gluconolactone Ki為1.1 μM,此抑制性質與不同來源的β-葡萄糖甘酵素相似。由氫核磁共振圖譜觀察酵素分解受質時,其產物為保留形態 (retention configuration) 的β-form葡萄糖。將酵素催化不同受質所得之 kcat 值以Bronsted plot分析所得圖形為向下彎曲的兩相圖,顯示其為兩步驟 (two-step) 之催化反應,包含glucosylation與 deglucosylation兩步驟,當受質為含有較佳離去基者 (pKa < 7.5,β1g = 0) ,其反應速率決定步驟為 deglucosylation step,有明顯的二級同位素效應kH/kD > 1.15,顯示其反應類似SN1機制,過渡狀態結構近似carboncation或其共振oxocarbonium ion。當受質為含有較差離去基者 (pKa > 7.5) ,得β1g = -0.85,其速率決定步驟應為 glucosylation step,而反應達過渡狀態時,已有大量電子轉移至離去基之氧上,推測glycosidic C─O鍵已顯著斷裂,其有二級同位素效應較弱 (kH/kD~1.05),故反應類似SN2機制,過渡狀態近似glucosyl-enzyme intermediate。
為進一步探討此酵素之親核性 (nucleophile) 與一般酸鹼催化基 (general acid/base),我們由family 3家族中的15個β-葡萄糖甘酵素成員做胺基酸序列比對及並以燕麥β-葡萄糖甘酵素之x-ray結構為模板進行分子模擬,結果顯示計有十一個胺基酸保留區如D71、R129、E132、E136、D137、K168、H169、D247、E177、D458、E473,經定點突變研究後,發現突變株的Km值除E473G外,其餘者皆變化不大,而kcat值除D247和E473G外,則下降10~3000倍。CD光譜研究顯示所有突變酵素並沒有明顯之二級結構變化。D247G和D247N的kcat/Km和wild-type相比下降了3×104和2×105倍,且經胺基酸序列的比對得知family 3家族“SDW”序列與fbgl “TD247Y”為高度保留區,由抑制劑conduritol-B-epoxide和wild type及D247E進行酵素失活反應 (irreversible inactivation),發現wild type會有明顯的不可逆失活現象 (ki = 0.014 s-1),而D247E之失活速率則相當緩慢,此結果顯示D247於催化反應中可能扮演親核基的角色。最後再利用活性位置不可逆抑制劑2’4’-dinitrophenyl-2-deoxy-2-fluoro-β-D-glucopyranoside (2F-DNPG) 標示作用及二次質譜法,由二次質譜分析結果定位出有標示糖基之短peptide片段的胺基酸序列為IVTD247YGINE,結果直接證明TD247Y是fbgl活性位置中的親核性基團。
十一個保留區位置中D71、R129、K168、H169、D247應位於活性區內,其餘保留區如E136、D137、E177、D275、D458、D469、E473亦均以定點突變方法研究之。其中突變株E136、D137、E177、D275、D458、D469的Km與wild type相較並無太大的差異,kcat值約為wild type 10%~80%,對於整個反應催化速率kcat/Km而言沒造成太大的影響,因此E136、D137、E177、D275、D458、D469雖被高度保留,但並非位於β-葡萄糖之活性催化區。相反的由活性之初步反應判斷E473可能為該酵素之酸鹼催化基。經深入之動力學研究,發現 (1) E473G與wide type之動力學數據比較,E473G對2,4-DNPG之 Km較wild type下降900倍,而kcat值下降3300倍,(2) E473G在pH 5.0~9.0之間kcat並沒有很大的變化,此與wild-type的bell-shape curve不一樣,表示E473突變後,general acid/base catalys已不存在,(3) E473G和2’4’-dinitrophenyl-β-D-glucopyranoside (2,4-DNPG) 反應之活性因添加azide而大幅增加,且生成β-glucosyl azide產生,(4) E473G與2-carboxyphenylβ-glucoside反應,其反應速率(kcat)和3-carboxyphenylβ-glucosides 及4-carboxyphenylβ-glucoside比較下,分別大60及107倍,且其對2-carboxyphenylβ-glucoside之反應性與wild type 相當,顯示E473G可藉由受質的酸基來回復活性 (5)利用活性位置不可逆抑制劑 N-bromoacetylglucosylamine (NBAGLN) 標示作用及二次質譜法分析,可確定E473為fbgl中的一般酸鹼催化胺基酸。
This study was focused on understanding the catalytic mechanism and identifying the nucleophile and general acid/base catalyst of the family 3 β-glucosidase from Flavobacterium meningosepticum.
Recombinant enzyme, fbgl, and mutants were purified from the crude extract of E. coli bearing correspondent genes. With the application of a SP cation-exchanged chromatography, enzymes can be obtained in good quality (> 90% homogeneity). Molecular weight (for all mutants) was analyzed by SDS-PAGE and shown to be ~80 kDa, which was consistent with that derived from DNA sequence. The wild type enzyme possessed highly specific activity on the glycone moiety, while it was relatively broad specificity towards the aglycone portion of the substrate. Its optimal activity was in pH 4.5~5.0. δ-gluconolactone was a competitively strong inhibitor (Ki = 1.1 μM) of the wild type enzyme. Glucose, however, exhibited a moderate product inhibition with Ki = 4.6 mM. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations including substrate reactivity, secondary kinetic isotope effect. The stereochemistry of the enzymatic hydrolysis was identified as occurring with the retention of anomeric configuration indicating a double-displacement reaction. Based on the kcat values of a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behavior was consistent with the two-step mechanism involving the formation and the breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (β1g = -0.85) for the leaving group-dependent portion (pKa of leaving phenols > 7.5) indicates a substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl, o-nitrophenyl, and p-cyanophenyl-β-D-glucopyanoside as substrates were 1.17 ±0.02, 1.19 ±0.02, and 1.04 ±0.02, respectively. These results supported an SN1-like mechanism for the deglucosylation step and an SN2-like mechanism for the glucosylation step.
Based on multi-alignment of amino acid sequences of fifteen enzymes from family 3, 11 highly conserved amino acids, including D71, R129, E132, E136, D137, E177, K168, H169, D247, D458 and E473, were studied by means of site-directed mutagenesis and kinetic investigations of the correspondent mutants. Results showed that, Km values of mutants were comparable to that of wild type (0.36 mM for DNPG), with the exception of E473G, Km =0.0004 mM. The kcat values were reduced some 10~3000-fold than that of wild type. The catalytic power of point mutation on D247 or E473 was crippled even more dramatically, e.g. kcat/Km of D247G and D247N were 3x104 and 2x105 times weaker than that of wild type, respectively. Yet, D247E mutant retained at least 20% activity of the wild type (WT) enzyme. Circular dichroism (CD) investigation revealed no significant differences among all mutants. Conduritol-B-epoxide, a potential active site-directed inhibitor, inactivated WT fbgl with a rate of 0.014 s-1, whereas a very slow rate was observed in the case of D247E mutant. These results strongly supported Asp-247 residue functions as the nucleophile of the catalytic reaction. A direct evidence was obtained from another active-site affinity labeling on WT by 2’,4’-dinitrophenyl-2-deoxy -2-fluoro-b-D-glucopyranoside (2F-DNPG) and following by tandem mass spectrometry analysis. The aspartate residue (D247) in the peptide of IVTDYTGINE was identified to be labeled.
On the basis of catalytic power analysis of all mutants, E473 residue was the best candidate of the general acid/base catalyst. Further detailed kinetic study confirmed this prediction shown as follows: (1) The kcat and Km value of E473G toward 2’4’-dinitrophenyl-β-D-glucopyranoside (2,4-DNPG) are reduced 3300-fold and 900-fold, respectively, in comparison with that of WT, (2) Unlike the bell-shaped pH profile of WT, the kcat values were virtually invariant with pH over the range of 5.0~9.0, indicating the general acid/base catalyst is absent on E473G mutant, (3) The activity of E473G towards 2,4-DNPG was largely enhanced by the addition of anion such as azide. b-Glucosyl azide was produced, (4) The catalytic activity of E473G towards 2-carboxyphenylβ-glucoside is comparable to that of WT and the correspondent kcat value (E473G) was 60 and 100-fold greater than those of 3-carboxyphenyl and 4-carboxyphenylβ-glucoside catalyzed by E473G, respectively. All of these results highly suggested E473 is the general acid/base catalyst, which was further confirmed by active-site affinity labeling of WT fbgl with N-bromoacetyl b-glucosylamine following by tandem mass spectrometry analysis. The glutamate (E473) in the peptide of SGESSSRANI was found to be labeled.

Flavobacterium meningosepticum中第三家族β-葡萄糖甘酵素的
反應機制研究及其親核性基團與一般酸鹼催化基團之鑑定
學生:池俊利 指導教授:李耀坤博士
國立交通大學應用化學研究所
摘 要
本研究旨在探討對自黃質菌 (Flavobacterium meningosepticum) 中選殖之family 3 β-葡萄糖甘酵素進行性質與反應機制之研究 (第三章) 及其親核性基團 (第四章 )與一般酸鹼催化基團 (第五章) 之鑑定。
基因重組及所有突變酵素均可由E.coli 細胞粗提取液經硫酸銨鹽沉澱,再以HiTrap SP陽離子交換樹脂管柱於 pH 6.9 下進行管柱層析分離而得純度可達 90 % 以上之酵素,其單體分子量約為79 kDa。以野生株 (wild type) 酵素而言,在可測量的範圍內,反應最佳之酸鹼值為4.5~5.0。此酵素對受質的特異性為糖基部份的選擇性甚嚴,但對非糖基部份的選擇性則較寬鬆。葡萄糖 (產物之一) 對此酵素有抑制效果,Ki 為4.6 mM,而δ-gluconolactone Ki為1.1 μM,此抑制性質與不同來源的β-葡萄糖甘酵素相似。由氫核磁共振圖譜觀察酵素分解受質時,其產物為保留形態 (retention configuration) 的β-form葡萄糖。將酵素催化不同受質所得之 kcat 值以Bronsted plot分析所得圖形為向下彎曲的兩相圖,顯示其為兩步驟 (two-step) 之催化反應,包含glucosylation與 deglucosylation兩步驟,當受質為含有較佳離去基者 (pKa < 7.5,β1g = 0) ,其反應速率決定步驟為 deglucosylation step,有明顯的二級同位素效應kH/kD > 1.15,顯示其反應類似SN1機制,過渡狀態結構近似carboncation或其共振oxocarbonium ion。當受質為含有較差離去基者 (pKa > 7.5) ,得β1g = -0.85,其速率決定步驟應為 glucosylation step,而反應達過渡狀態時,已有大量電子轉移至離去基之氧上,推測glycosidic C─O鍵已顯著斷裂,其有二級同位素效應較弱 (kH/kD~1.05),故反應類似SN2機制,過渡狀態近似glucosyl-enzyme intermediate。
為進一步探討此酵素之親核性 (nucleophile) 與一般酸鹼催化基 (general acid/base),我們由family 3家族中的15個β-葡萄糖甘酵素成員做胺基酸序列比對及並以燕麥β-葡萄糖甘酵素之x-ray結構為模板進行分子模擬,結果顯示計有十一個胺基酸保留區如D71、R129、E132、E136、D137、K168、H169、D247、E177、D458、E473,經定點突變研究後,發現突變株的Km值除E473G外,其餘者皆變化不大,而kcat值除D247和E473G外,則下降10~3000倍。CD光譜研究顯示所有突變酵素並沒有明顯之二級結構變化。D247G和D247N的kcat/Km和wild-type相比下降了3×104和2×105倍,且經胺基酸序列的比對得知family 3家族“SDW”序列與fbgl “TD247Y”為高度保留區,由抑制劑conduritol-B-epoxide和wild type及D247E進行酵素失活反應 (irreversible inactivation),發現wild type會有明顯的不可逆失活現象 (ki = 0.014 s-1),而D247E之失活速率則相當緩慢,此結果顯示D247於催化反應中可能扮演親核基的角色。最後再利用活性位置不可逆抑制劑2’4’-dinitrophenyl-2-deoxy-2-fluoro-β-D-glucopyranoside (2F-DNPG) 標示作用及二次質譜法,由二次質譜分析結果定位出有標示糖基之短peptide片段的胺基酸序列為IVTD247YGINE,結果直接證明TD247Y是fbgl活性位置中的親核性基團。
十一個保留區位置中D71、R129、K168、H169、D247應位於活性區內,其餘保留區如E136、D137、E177、D275、D458、D469、E473亦均以定點突變方法研究之。其中突變株E136、D137、E177、D275、D458、D469的Km與wild type相較並無太大的差異,kcat值約為wild type 10%~80%,對於整個反應催化速率kcat/Km而言沒造成太大的影響,因此E136、D137、E177、D275、D458、D469雖被高度保留,但並非位於β-葡萄糖之活性催化區。相反的由活性之初步反應判斷E473可能為該酵素之酸鹼催化基。經深入之動力學研究,發現 (1) E473G與wide type之動力學數據比較,E473G對2,4-DNPG之 Km較wild type下降900倍,而kcat值下降3300倍,(2) E473G在pH 5.0~9.0之間kcat並沒有很大的變化,此與wild-type的bell-shape curve不一樣,表示E473突變後,general acid/base catalys已不存在,(3) E473G和2’4’-dinitrophenyl-β-D-glucopyranoside (2,4-DNPG) 反應之活性因添加azide而大幅增加,且生成β-glucosyl azide產生,(4) E473G與2-carboxyphenylβ-glucoside反應,其反應速率(kcat)和3-carboxyphenylβ-glucosides 及4-carboxyphenylβ-glucoside比較下,分別大60及107倍,且其對2-carboxyphenylβ-glucoside之反應性與wild type 相當,顯示E473G可藉由受質的酸基來回復活性 (5)利用活性位置不可逆抑制劑 N-bromoacetylglucosylamine (NBAGLN) 標示作用及二次質譜法分析,可確定E473為fbgl中的一般酸鹼催化胺基酸。
Mechanistic study and direct evidence on identification of the nucleophile and acid/base catalyst of a family 3 β-glucosidase from Flavobacterium meningosepticum
Student:Jiunly Chir Advisor:Dr. Yaw-Kuen Li
Department of Applied Chemistry
National Chiao Tung University
Abstract
This study was focused on understanding the catalytic mechanism and identifying the nucleophile and general acid/base catalyst of the family 3 β-glucosidase from Flavobacterium meningosepticum.
Recombinant enzyme, fbgl, and mutants were purified from the crude extract of E. coli bearing correspondent genes. With the application of a SP cation-exchanged chromatography, enzymes can be obtained in good quality (> 90% homogeneity). Molecular weight (for all mutants) was analyzed by SDS-PAGE and shown to be ~80 kDa, which was consistent with that derived from DNA sequence. The wild type enzyme possessed highly specific activity on the glycone moiety, while it was relatively broad specificity towards the aglycone portion of the substrate. Its optimal activity was in pH 4.5~5.0. δ-gluconolactone was a competitively strong inhibitor (Ki = 1.1 μM) of the wild type enzyme. Glucose, however, exhibited a moderate product inhibition with Ki = 4.6 mM. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations including substrate reactivity, secondary kinetic isotope effect. The stereochemistry of the enzymatic hydrolysis was identified as occurring with the retention of anomeric configuration indicating a double-displacement reaction. Based on the kcat values of a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behavior was consistent with the two-step mechanism involving the formation and the breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (β1g = -0.85) for the leaving group-dependent portion (pKa of leaving phenols > 7.5) indicates a substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl, o-nitrophenyl, and p-cyanophenyl-β-D-glucopyanoside as substrates were 1.17 ±0.02, 1.19 ±0.02, and 1.04 ±0.02, respectively. These results supported an SN1-like mechanism for the deglucosylation step and an SN2-like mechanism for the glucosylation step.
Based on multi-alignment of amino acid sequences of fifteen enzymes from family 3, 11 highly conserved amino acids, including D71, R129, E132, E136, D137, E177, K168, H169, D247, D458 and E473, were studied by means of site-directed mutagenesis and kinetic investigations of the correspondent mutants. Results showed that, Km values of mutants were comparable to that of wild type (0.36 mM for DNPG), with the exception of E473G, Km =0.0004 mM. The kcat values were reduced some 10~3000-fold than that of wild type. The catalytic power of point mutation on D247 or E473 was crippled even more dramatically, e.g. kcat/Km of D247G and D247N were 3x104 and 2x105 times weaker than that of wild type, respectively. Yet, D247E mutant retained at least 20% activity of the wild type (WT) enzyme. Circular dichroism (CD) investigation revealed no significant differences among all mutants. Conduritol-B-epoxide, a potential active site-directed inhibitor, inactivated WT fbgl with a rate of 0.014 s-1, whereas a very slow rate was observed in the case of D247E mutant. These results strongly supported Asp-247 residue functions as the nucleophile of the catalytic reaction. A direct evidence was obtained from another active-site affinity labeling on WT by 2’,4’-dinitrophenyl-2-deoxy -2-fluoro-b-D-glucopyranoside (2F-DNPG) and following by tandem mass spectrometry analysis. The aspartate residue (D247) in the peptide of IVTDYTGINE was identified to be labeled.
On the basis of catalytic power analysis of all mutants, E473 residue was the best candidate of the general acid/base catalyst. Further detailed kinetic study confirmed this prediction shown as follows: (1) The kcat and Km value of E473G toward 2’4’-dinitrophenyl-β-D-glucopyranoside (2,4-DNPG) are reduced 3300-fold and 900-fold, respectively, in comparison with that of WT, (2) Unlike the bell-shaped pH profile of WT, the kcat values were virtually invariant with pH over the range of 5.0~9.0, indicating the general acid/base catalyst is absent on E473G mutant, (3) The activity of E473G towards 2,4-DNPG was largely enhanced by the addition of anion such as azide. b-Glucosyl azide was produced, (4) The catalytic activity of E473G towards 2-carboxyphenylβ-glucoside is comparable to that of WT and the correspondent kcat value (E473G) was 60 and 100-fold greater than those of 3-carboxyphenyl and 4-carboxyphenylβ-glucoside catalyzed by E473G, respectively. All of these results highly suggested E473 is the general acid/base catalyst, which was further confirmed by active-site affinity labeling of WT fbgl with N-bromoacetyl b-glucosylamine following by tandem mass spectrometry analysis. The glutamate (E473) in the peptide of SGESSSRANI was found to be labeled.
謝 誌
衷心感謝吾師 李耀坤博士,於這五年來在課業上及實驗上的殷切指導與幫助,使我對理論或是實作技術上給予最完整的指導。也要感謝 蕭介夫、陳水田、林立元、黃鎮剛四位口試委員對論文內容給予的指導。
研究的生涯是艱苦的,但慶幸有實驗室的所有伙伴們,至玉、金鳳、彥杰、及各位學弟妹們於口試這段期間的大力幫忙,也謝謝你們陪伴我度過這段研究所的歲月。
還要感謝在我低潮期,給我加油與鼓勵的安台,因為你的包容及關懷使我更加有勇氣與毅力面對每一個難關,最後僅將這一份的成果獻給家中的親人,謝謝你們這些年來的關心與支持這是我一路走來心中最大的支柱。
目錄
中文摘要i
英文摘要v
誌謝ix
目錄x
圖目錄xvi
表目錄xx
簡寫表xxii
第一章、緒論1
1-1 纖維素分解酵素群 (cellulases)1
1-2 β-葡萄醣甘酵素 (β-glucosidase)3
1-3醣類水解酵素的分類法4
1-4 β-葡萄糖甘酵素研究 7
1-5 β- glucosidase的純化與選殖22
1-6 本論文的研究動機22
第二章、實驗方法24
2-1 定點突變24
2-1-1設計原理24
2-1-2定點突變操作步驟25
2-1-3 Competent cell的製備26
2-1-4轉型competent cell26
2-1-5 Cycle sequencing步驟27
2-2 選殖及定點突變β-葡萄糖甘酵素的純化28
2-2-1一般敘述28
2-2-2 胞內粗提取液的取得與硫酸銨沈澱29
2-2-3 HiTrap SP陽離子交換樹脂管柱層析30
2-3 芳香類-β-D-葡萄糖甘化合物(aryl-β-D-glucopyranoside)
的合成31
2-4 β-葡萄糖甘酵素的特性研究32
2-4-1 酸鹼度對wild typeβ-葡萄糖甘酵素活性的影響32
2-4-2 抑制劑與酵素解離常數的測定32
2-4-3氫核磁共振光譜研究β-葡萄糖甘酵素酵素的立體
選擇性33
2-4-4 β-葡萄糖甘酵素的受質反應專一性實驗33
2-4-4-1 改變受質之糖基部分33
2-4-4-2 改變受質之非糖基部分34
2-4-5 D71N酸鹼度對酵素活性的影響35
2-4-6 失活劑的研究35
2-4-7 親和性活性位置標的物(包含胃蛋白酵素消化
作用)位與串聯質譜的定位作用36
2-4-8 酸鹼度對突變酵素(E473Q、E473D、E473G)活性
的影響37
2-4-9 陰離子親核劑對酵素活性的影響38
2-4-10利用1H NMR觀測azide反應後之產物-疊氮
葡萄糖β-glucosyl azide39
2-4-11 受質反應專一性實驗39
2-4-12 β-葡萄糖甘酵素受質協助催化
(substrate-assisted catalysis) 水解反應40
2-4-13 CD (circular dichroism)光譜之研究40
第三章、反應機構之研究41
3-1β-葡萄糖甘酵素的純化42
3-2芳香類-β-D-葡萄糖化合物(aryl-β-D-glucopyranosides
的合成)44
3-3β-葡萄糖甘酵的催化性質討論45
3-3-1酸鹼度對酵素活性的影響45
3-3-2抑制作用的研究47
3-3-2-1 競爭性抑制作用50
3-3-3受質特異性研究53
3-3-4反應機構的研究57
3-3-4-1 氫核磁共振儀研究β-葡萄糖甘酵素的立
體選擇性58
3-3-4-2 Bronsted polt62
3-3-4-3二級同位素效應研究64
3-4結論69
第四章 鑑定β-葡萄糖甘酵素活性位置中扮演親核性
(nucleophlic attack)基團 71
4-0 前言72
4-1 β-葡萄糖甘酵素親核性基團之鑑定75
4-2 β-葡萄糖甘酵素突變株之純化及活性表建立77
4-2-1 β-葡萄糖甘酵素突變株之二級結構與Km關係之探討79
4-2-2 β-葡萄糖甘酵素各個突變株性質之討論80
4-2-2-1 E136及D137突變株性質之討論80
4-2-2-2 D71突變株性質之討論81
4-2-2-3 R129突變株性質之討論82
4-2-2-4 H169及K168突變株性質之討論83
4-2-2-5 E132突變株性質之討論85
4-3 β-葡萄糖甘酵素活性位置中 D247為親核性基團之鑑定方式85
4-3-1 D247胺基酸序列比對85
4-3-2 D247與抑制劑研究87
4-3-3 親和性活性位置標的物與串聯質譜的定位作用
(active site affinity labelling and tandem mass
spectrometric localization)90
4-4 結論98
第五章、 鑑定β-葡萄糖甘酵素活性位置中一般酸鹼催
化(general acid/base)之胺基酸100
5-1一般酸鹼催化基團胺基酸的鑑定方法101
5-2 β-葡萄糖甘酵素E473突變株酸鹼度對活性的研究106
5-3 不同陰離子親核劑對E373突變株酵素之反應109
5-4 Azide對酵素反應之影響113
5-5 E373突變點之Bronsted plot 探討116
5-6 β-葡萄糖甘酵素與受質協助催化(substrate-assisted
catalysis)之水解反應121
5-7以質譜儀鑑定E473為fbgl之一般酸鹼催化基團126
5-8親和性活性位置標的物與串聯質譜的定位作用
(active site affinitylabelling and tandem mass
spectrometric localization)127
5-9結論135
第六章 總結論138
第七章 參考文獻138
附圖……………………………………………………………………145
圖目錄
圖1 植物纖維素的構造1
圖2纖維素分解酵素群的協力作用2
圖3糖甘的水解反應3
圖4 Abg 及Asp突變株與失活劑之反應圖8
圖5 突變株Glu358Asp酵素與過量的2F-DNPG隨時間
變化的反應圖9
圖6 失活的Abg 在沒加 (●) 及有加 (■) β-D- glucosyl
benzene的復活圖形9
圖7 Abg 與Asp mutant的反應能量圖13
圖8 糖類水解酵素常用的失活劑結構圖15
圖9 親和性標示物與串聯質譜的定位流程圖18
圖10為Abg之化學復活方法(rescure)的機制圖20
圖11 Abg中受質分子內酸協助催化示意圖21
圖12 Wild type (fbgl) HiTrap SP 陽離子交換樹脂層析結果圖43
圖13 HiTrap SP 陽離子交換樹脂層析圖結果之SDS-PAGE圖44
圖14反應步驟之簡圖45
圖15以4-chloro-2-nitrophenyl-β-D- glucoside 為受質時酸
鹼值對酵素活性的影響46
圖16以PNPG為受質時酸鹼值對酵素活性的影響47
圖17 Glucose的Ki值52
圖18β-葡萄糖甘酵素可能的反應機構簡圖57
圖19酵素反應中糖基C1上氫原子之化學位移變化59
圖20以氫核磁共振儀(NMR)追縱酵素反應立體選擇性60
圖21 Bronsted Plot log rkcat 對 pKa 作圖63
圖22 Bronsted Plot log rkcat / Km 對 pKa 作圖63
圖23β-葡萄糖甘酵素 (fbgl) 的催化反應之能量圖66
圖24β-葡萄糖甘酵素 (fbgl) 的反應機構及其過渡狀態
之結構67
圖25β-葡萄糖甘酵素之序列比對73
圖26燕麥β-葡萄糖甘酵素活性中心位置的胺基酸74
圖27燕麥與F. meningosepticum兩者酵素胺基酸序列比對75
圖28 Wild type 與突變株酵素之CD光譜比較79
圖29 Wild type 與D71N pH profile 之比較81
圖30 Wild type (X) 與突變株D247N (○)、D247E (●)之
CD圖譜比較85
圖31 Wild type 及D247E (8.5 mM CBE) 在不同CBE濃度
的殘留活性區線圖87
圖32 Wild type與2F-DNPG的反應圖90
圖33 Wild typeβ-glucosidase的質譜圖93
圖34 Wild typeβ-glucosidase與2F-DNPG作用90分鐘
後的質譜圖93
圖35經由RP-HPLC與2F-DNPG標示過的peptide片段之
層析圖 94
圖36將上圖由RP-HPLC分離出的peptide片段進行二次質
譜的分析 (Tandem mass analysis) 所得之圖譜圖96
圖37Wild-type 酸鹼度對活性的影響106
圖38突變株E473G酸鹼度對活性的影響106
圖39突變株E473Q酸鹼度對活性的影響107
圖40E473D酸鹼度對活性的影響107
圖41 E473G與DNPG之氫核磁共振圖譜111
圖42 Azide 於E473G 反應中可能扮演的角色112
圖43 E473G之kcat相對不同濃度的azide對不同aryl
glucosides的酵素水解反應114
圖44 Bronsted plot:E473G對具有不同離去基能力的受
質水解之相對速率圖117
圖45 Bronsted plot: E473D對具有不同離去基能力的
受質水解之相對速率圖118
圖46受質協助催化的示意圖122
圖47 E473G 與DNPG 反應5分鐘後的質譜圖125
圖48 Wild type與NBAGLN的反應圖126
圖49 Wild type 與N-bromoacetyl-β-D-glucosylamine反應
90分後的質譜圖。129
圖50經由RP-HPLC將與NBAGLN標示過的胜肰片段之層
析圖130
圖51將上圖由RP-HPLC分離出的peptide片段之二次質譜
分析 (Tandem mass analysis) 之圖譜圖131
圖52經由trypsin行消化水解而得到之片段,再將之進行二
次質譜分析之圖譜圖132
圖53 經LysineC行消化水解而得到之片段,再將之進行二
次質譜分析133
表目錄
表1 從數種來源不同的β-葡萄糖甘酵素對glucose及δ-gluconolactone之抑制性質比較53
表2 β-葡萄糖甘酵素(fbgl)對各種糖甘化合物之Km及rkcat值55
表3 β-葡萄糖甘酵素(fbgl)對葡萄糖甘化合物之Km及r kcat值62
表4 β-葡萄糖甘酵素 (fbgl) 的二級同位素效應64
表5 突變株與wild type活性之比較77
表6 突變株與wild type活性之比較77
表7 Wild type 與D247突變株活性之比較86
表8 Wild type 與突變株的抑制常數 (Ki)表88
表9 8段序列於質譜儀中帶單位正電荷的陽離子之peptide 92
表10突變株與wild type 的比較104
表11突變株與wild type 的比較104
表12不同之親核性陰離子的存在下對2,4-DNPG之酵素
水解反應109
表13 E473G對於不同離去基之受質反應之Km及kcat115
表14 E473G在200 mM azide下對於不同離去基之受質反應
之Km及kcat115
表15 E473D對於不同離去基之受質反應之Km及kcat116
表16 E473G在azide的存在與否下和不同取代位置的
carboxyphenylβ-D-glucoside反應之活性比較表122
表17 8段序列於質譜儀中帶單位正電荷的陽離子之peptide128
簡寫:
PNPX, p-nitrophenyl-b-D-xylopyranoside;
PNPA, p-nitrophenyl-a-L-arabinopyranoside;
PNPGal, p-nitrophenyl-b-D-galactopyranoside;
PNPAG, p-nitrophenyl-b-D-N-acetylglucosaminide;
PNPM, p-nitrophenyl-b-D-mannopyranoside,
PG, phenyl-b-D-glucopyranoside;
ONPG, o-nitrophenyl-b-D-glucopyranoside;
PNPG, p-nitrophenyl-b-D-glucopyranoside;
PCPG, p-cyanophenyl-b-D-glucopyranoside;
CNPG, 4-chloro-2-nitrophenyl-b-D-glucopyranoside;
DNPG, 2,4-dinitrophenyl-b-D-glucopyranoside;
3,4-DNPG, 3,4-dinitrophenyl-b-D-glucopyranoside;
2,5-DNPG, 2,5-dinitrophenyl-b-D-glucopyranoside;
2-CPG,2-carboxy-pheny-b-D-glucoside;
3-CPG,3-carboxy-pheny-b-D-glucoside;
4-CPG,4-carboxy-pheny-b-D-glucoside;
2-CEPG,2-carboxyester-phenyl-b-D-glucoside;
4-CEPG,4-carboxyester-phenyl-b-D-glucoside;
2F-DNPG,2’4’-dinitrophenyl-2-deoxy-2-fluoro-b-D-glucopyranoside;
NBAGLN;N-bromoacetylglucosylamine
NAGLN, N-acetyl-glucosylamine;
ESI, electrospray ioniza-tion;
RP-HPLC, reverse phasehigh-performance liquid chromatography;
MS, mass spectrometry;
MS/MS, tandem mass spectrometry;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
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