Mechanistic study and direct evidence on identification of the nucleophile and acid/base catalyst of a family 3 β-glucosidase from Flavobacterium meningosepticum
|關鍵字:||β-葡萄糖甘酵素;親核基;一般酸鹼催化之胺基酸;機制;β-glucosidase;Nucleophile residue;general acid/base catalyst residue;mechanism|
|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。|
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.