單核心與雙核心金屬結合位置的比較：從PDB資料庫分析與DFT/CDM計算說明金屬對蛋白質活化位置的親合性與選擇性

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研究生：	楊宗穎 Tzung Yin Yang
論文名稱：	單核心與雙核心金屬結合位置的比較：從PDB資料庫分析與DFT/CDM計算說明金屬對蛋白質活化位置的親合性與選擇性 Mononuclear versus Binuclear Metal-Binding Sites: Metal Binding Affinity and Selectivity from PDB Survey and DFT/CDM Calculations
指導教授：	林小喬 Carmay Lim
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	30
中文關鍵詞：	鎂、鋅、金屬蛋白
外文關鍵詞：	magnesium, zinc, metalloprotein, homonuclear, trinuclear, bridging ligand
相關次數：	點閱：3 下載：0
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雙核心金屬中心在含金屬酵素反應中常參與水解、水合、異構化以及氧化還原等步驟。儘管目前對此類型蛋白質結構、功能特性等研究與日遽增。然而，蛋白質之於金屬的選擇性與親合性仍有許多尚未解決的問題：(1)哪種金屬以及其形態最常出現於含金屬蛋白質中？金屬的間距是否有一固定範圍？哪種氨基酸最易與兩個金屬同時將接？以及哪些氨基酸較易成為第一層結構組成？(2)於雙金屬核心中，其金屬之間對於彼此於活化位置的親合性與選擇性是否有影響？(3)蛋白質活化位置對於金屬的親合性與選擇性於雙金屬核心和單心屬核心中是否有所不同？在此，吾人將結合Protein Data Bank資料庫搜尋以及DFT/CDM計算以解決上述問題。以資料庫搜尋了解最常見的橋樑配基(bridging ligands)與非橋樑配基(non-bridging ligands)；從計算中，依據(i)配基組成和結合位置的電荷價數；(ii)金屬離子形態與配位數；(iii)結合位置於溶劑的裸露情況以及本身架構的柔韌性等條件計算鎂離子跟鋅離子於雙金屬結合位置的自由能。研究結果指出同形態雙金屬核心中，金屬依照其金屬價荷數以及電子接受能力分類，以及Asp/Glu是兩個最常見的橋樑配基。亦指出若鎂離子佔據雙金屬核心的其中一個位置後，將狹隘另一位置的金屬選擇性卻提高其選擇性。以上結果皆符合實驗上所發現的現象。

Binuclear metal centers in metalloenzymes are involved in a number of hydrolytic, hydration, isomerization, and redox processes. Despite the growing number of studies elucidating their structure, properties, and function, questions regarding certain aspects of the bimetallic proteins’ biochemistry still remain; e.g., (i) What are the general characteristics of binuclear sites found in 3D structures such as the range of metal訃metal distances, and the most common ligand bridging the two metal cations? (ii) How does the presence of a metal cation in one of the binuclear sites affect the metal訃binding affinity/selectivity of the other site? (iii) How do the characteristics and metal訃binding affinity/selectivity of binuclear sites compare with those of their mononuclear counterparts? Here we address these questions by combining a Protein Data Bank survey of binuclear sites with density functional theory (DFT) combined with continuum dielectric method (CDM) calculations. The results reveal that for homo訃binuclear sites, the metal separation depends on the metal’s charge and the electron-accepting ability, and Asp-/Glu-, bidentately bound to the two cations, is the most common bridging ligand. They also reveal that Mg2+洶occupying one of the binuclear sites attenuates the metal訃binding affinity, but enhances the selectivity of its neighboring site, compared to the corresponding mononuclear counterparts. These findings are consistent with available experimental data. The weak metal binding of one of the binuclear sites would enhance the metal cofactor mobility in achieving the transition state, whereas the enhanced selectivity of Mg2+訃Mg2+ centers help protect against unwanted substitutions by transition metal ions, which are generally stronger Lewis acids compared to Mg2+.

Contents
摘要...............…………………………………………………………….................    i
Abstract………………………………….…………………………………….........   ii
Contents…………………………..…………………………………………………  iii
List of Figures and Table……………………………...………………………….....   v

1 Introduction…………………………………………………………………………………..    1
2 Methods……………………………………………………………………………………….    4
    2.1 Database Survey……………………………………………………………………..     4
    2.2 Defining First-shell Ligands in the Binuclear Metal-binding Sites……………...     4
    2.3 Models Used………………………………………………………………………….     4
    2.4 DFT Calculations…………………………………………………………………….     5
        2.4.1 Geometries…………………………………………………………………..     5
        2.4.2 Gas–phase Free Energies…………………………………………………...    5
    2.5 Continuum Dielectric Calculations………………………………………………....     5
3 Results…………………………………………………………………………………………    7
    3.1 PDB Survey…………………………………………………………………………...    7
    3.2 Metal binding affinities in mononuclear and binuclear binding sites…………….    9
    3.3 Metal cation selectivity in mononuclear and binuclear binding sites……………..  10
        3.3.1 Rigid Sites……………………………………………………………………  11
        3.3.2 Flexible Sites………………………………..……………………………….. 11
4 Discussion……………………..……………..……………..……………..……………..….. 13
    4.1 Effect on Metal–Binding Affinity……………………………………………………  13
        4.1.1 Experimental Support………………………………………………………..  14
    4.2 Effect on Metal–Binding Selectivity………………………………………………… 14
4.3 Biological Implications……………………………………………………………….  15
5 REFERENCES............................................................................................................... 16

List of Figures and Table
Figures
Figure 1. Plot of the average inter-metallic bond distance in homo-binuclear binding sites, <M-M>, with respect to the metal charge, qM. The Pearson correlation coefficient is 0.76……… 19
Figure 2. Percentage frequency distribution of 1st-shell ligands observed in the PDB structures of proteins containing homo-binuclear binding sites. The grey and black bars represent the % frequency distributions of the bridging and non-bridging ligands, respectively…………………..  20
Figure 3. Comparison between the percentage frequency distributions of 1st-shell ligands in mononuclear and homo-binuclear PDB sites. The blue and red bars represent the % frequencies observed in homo-binuclear PDB sites (this work) and mononuclear PDB sites (from Dudev et al.42), respectively.……………… ……………………………………………………………………….  24
Figure 4. Free energies, ΔGx (in kcal/mol), for replacing H2O with CH3COO- (ACE-) and [(CH?3COO) Mg (H2O)3 (CH3COO)2]- (denoted by [Mg-SL]-) in (a) [Mg (H2O)6]2+, (b) [Mg (H2O)5 CH3COO]+, and (c) [Mg (H2O)4 (CH3COO)2]0 complexes in a protein cavity characterized by dielectric constant, x……………………………………………………………………………….. 26
Figure 5. Free energies, ΔGx (in kcal/mol), for replacing Mg2+ with Zn2+ in rigid (a) neutral and (b) anionic mono and binuclear Mg2+-binding sites characterized by dielectric constant, x………….  27
Figure 6. Free energies, ΔGx (in kcal/mol), for replacing Mg2+ with Zn2+ in flexible (a) neutral and (b) anionic mono and binuclear Mg2+-binding sites characterized by dielectric constant, x……...  28
Figure 7. Free energies, ΔGx (in kcal/mol), for replacing Mg2+ with Zn2+ in a flexible trinuclear Mg2+-binding site characterized by dielectric constant, x…………………………………………  29

Table
Table 1. Average distances (in ?) between the two metal ions in homo-binuclear binding sites……………………………………………………………………………………………….  30

                                

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