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研究生:蘇偉廷
論文名稱:二價金屬離子對胃幽門螺旋桿菌之菸鹼醯胺腺嘌呤雙核苷酸-啶黃素氧化還原酵素(HP0642)的影響
論文名稱(外文):The effects of divalent metal ions on oxidoreduction and nitroreduction of the NAD(P)H:flavin oxidoreductase (HP0642) from Helicobacter pylori
指導教授:黃海美
指導教授(外文):Huang, Haimei
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
論文頁數:60
中文關鍵詞:菸鹼醯胺腺嘌呤雙核苷酸-啶黃素氧化還原酶硝基還原酶
外文關鍵詞:HP0642NAD(P)H:flavin oxidoreductasenitroreductasenitrofuranzonenitrofurantoin
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在先前的研究當中指出由大腸桿菌(E. coli)SG13009表現系統所獲得之重組HP0642蛋白(楊瑞榮 碩士論文, 2006)有兩個主要的功能:一是作為啶黃素氧化還原酵素( NAD(P)H-flavin oxidoreductase ),依賴菸鹼醯胺腺嘌呤雙核苷酸(磷酸)[NAD(P)H]為電子提供者,可還原酵素蛋白上所結合的啶啶黃素(Flavin),二是作為硝基還原還原酵素 (nitroreductase),以相同的電子提供者─可還原硝基化物(鍾榮育碩士論文, 2007)。此外,在之前的研究當中已發現1 mM Zn+2或Hg+2可以抑制NADH作為電子提供者─啶黃素氧化還原酵素;當反應中有5 mM of Zn+2或Fe+2 NADPH作為電子提供者的硝基還原酵素被抑制,Cu+2 則使相同酵素作用增加(楊瑞榮 碩士論文, 2006)。

本研究裡重組HP0642蛋白之啶黃素氧化還原酵素活性是以反應中NADH氧化為NAD+時使波長340 nm吸光值減少,作為量測。啶黃素氧化還原活性的Km值為4.3 μM,Vmax值為303.00 μM min-1 mg-1 (Fig. 1)。

分別加入以下七種金屬離子鎂、鈣、錳、鈷、鎳、銅、和鋅,檢查其對重組HP0642蛋白之NADH─啶黃素氧化還原酵素活性是否有影響。結果是相對於實驗的控制組,在1.6 mM濃度的鋅、錳、鎂離子分別抑制酵素活性39.39、74.18 和79.22%。鈣、鈷、鎳、銅在0.2 ~1.6 mM範圍裡,並沒有很明顯的抑制現象。而在相對高濃度2~16 mM範圍裡,銅、錳、和鈷離子可抑制氧化還原活性至少到達50%。在16 mM時,相對控制組而言,金屬離子抑制後,所剩餘之氧化還原活性為銅離子 (15.07%)、錳離子 (41.83%)、鈷離子 (48.96%)、鋅離子 (55.73%)、鎳離子 (62.50%)、鎂離子 (73.37%)、和鈣離子 (97.78%) (Fig. 2)。

重組HP0642蛋白之硝基還原酵素活性是以反應中加入的含氮化物如呋喃西林類(nitrofurazone, NFZ or nitrofuratoin, NFT)之NO官能基氧化為 -NHOH官能基使波長400 (NFZ) 或420 (NFT) nm吸光值減少,作為量測。呋喃西林NFZ還原活性的Km值為161.29 μM,Vmax值為1428.57 μM min-1 mg-1 (Fig. 3)。呋喃西林(NFT)還原活性的Km值為66.73 μM,Vmax值為384.62 μM min-1 mg-1 (Fig. 7)。而上述同樣的金屬離子也分別用來觀察對硝基還原活性的影響。實驗發現NFZ參與的酵素反應裡分別加入0.2 mM鎂、鈣、錳、鈷、和鎳離子能夠分別增加硝基還原活性到107.54 %、111.51 %、117.06 %、108.73 %和122.62 %。而銅離子在這個濃度則是會抑制活性,和控制組相比為64.68 %。鋅離子在硝基還原活性的測試當中,和控制組相比為93.65 % (Fig. 4),可說相差無幾,和前面所發現會抑制氧化還原活性的特性無關,未來需要更進一步探討。

實驗發現鎂和鈣依然可以稍為增加還原活性。鈷、錳、鎳和鋅離子抑制程度 分別為:3, 4, 14, 24%。相同的濃度0.2 mM銅離子,則完全抑制了呋喃妥因(NFT)還原活性 (Fig. 8)。銅離子的濃度降低十倍到0.02 mM,仍可使重組HP0642蛋白之硝基還原酵素活性降低到80.69 % ( Fig. 9)。

銅離子對呋喃西林(NFZ)與呋喃妥因(NFT)還原的抑制作用更進一步作用機制初步的研究結果,可以由其動力飽合曲線(Kinetics, Saturation curve)之Lineweaver–Burk雙倒數圖結果來作一結論:兩者均接近un-competitive inhibition (Fig. 6) (Fig. 10)。

In the previous study, the recombinant HP0642 protein which obtained from gene expression in E. coli SG13009 system has two majority functions as NAD(P)H electron donor dependent NAD(P)H-flavin oxidoreductase and the same electron donor dependent nitroreductase. In the previous study, it was found that the oxidoreductase activity of recHP0642 was inhibited by 1 mM Zn2+ or Hg2+ when NADH as electron donor. And the nitroreductase activity of recHP0642 was inhibited by 5 mM Zn2+ and Fe2+ when NADPH as electron donor. But the Cu2+ could make the nitroreductase activity increase.

The NADH:flavin oxidoreductase activity could be monitored by the decrease of the absorbance of NADH at 340 nm. The Km value is 4.3 μM, the Vmax is 303.00 μM min-1 mg-1 in this study (Fig. 1).

The divalent metal ion, including Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+, were used to observe the effect on oxidoreductase activity for recHP0642 protein. The Mg2+, Mn2+, and Zn2+ at 1.6 mM could inhibit the activity down to 79.22, 74.18, and 39.39% relative to metal-free control group. Among the range at 0.2~1.6 mM, the Ca2+, Co2+, Ni2+, and Cu2+ performed no obvious effects on the activity. At the relative high concentration 2~16 mM, Cu2+, Mn2+, and Co2+ could inhibit the activity down to 50% relative to metal-free control group at least. At 16 mM, the remaining activity order of oxidoreductase of HP0642 protein is as follows: Cu2+ (15.07%), Mn2+ (41.83%), Co2+ (48.96%), Zn2+ (55.73%), Ni2+ (62.50%), Mg2+ (73.37%), and Ca2+ (97.78%) (Fig. 2).

The nitroreductase activity of HP0642 protein was monitored by the decrease of the absorbance of Nitrofurazone and Nitrofuratoin at 400 or 420 nm respectively. The Km value is 161.29 μM, the Vmax is 1428.57 μM min-1 μg-1, and the kcat value is 600.00 sec-1 in Nitrofurazone nitroreductase activity (Fig 3). The Km value is 66.73 μM, the Vmax is 384.62 μM min-1 μg-1, and the kcat value is 164.54 sec-1 in nitrofurantoin nitroreductase activity (Fig 7). The divalent metal ions which mentioned above were used to observe the nitroreductase activity for recHP0642 protein. Mg2+, Ca2+, Mn2+, Co2+, and Ni2+ at 0.2 mM could change the activity up to 107.54 %, 111.51 %, 117.06 %, 108.73 %, and 122.62 % of metal-free control level. Another tested divalent cation, Cu2+, could inhibit enzyme activity down to 64.68 % of metal-free control level at the same concentration. Zn2+ with slightly inhibition effect on nitroreductase activity, 93.65 % of metal-free control level was observed (Fig. 4). The Zn2+ showed different properties in reaction of oxidoreductase and nitroreductase activity of recHP0642, and the phenomenon should be discussed detailed in the future.

Mg2+, and Ca2+ could make the nitrofurantoin nitroreductase activity up to 104.52 %, 104.40 % of metal-free control level. Mn2+, Co2+, Ni2+ and Zn2+ could be with interfered effect on nitroreductase activity down to 95.96 %, 96.08 %, 86.03% and 76.73% of metal-free control level. The Cu2+ could inhibit the nitrofuratoin nitroreductase activity completely at 0.2 mM (Fig. 8). According to this result, the concentration of Cu2+ cation was diluted 10 times to 0.02 mM. The activity was inhibited by Cu2+ down to 80.69 % of metal-free control level (Fig 9).

In this study, both the reduction of nitrofurazone and nitrofuratoin was inhibited by Cu2+ with decreasing Vmax and the Km value, and it is concluded that the inhibition type of Cu2+ on nitroreductase activity of recHP0642 preotein was uncompetitive inhibition ( Fig. 5, 10)(Fig. 6, 11).

摘要 5
Abstract 7
Introduction 9
1. Helicobacter pylori 9
2. NAD(P)H:flavin oxidoreductase 10
3. The physiological importance of NAD(P)H:Flavin Oxidoreductase 11
4. The background of nitrofurazone and nitrofurantoin 11
5. Multi-substrate reactions 12
(A) Ternary-complex mechanisms (appendix 4A) 13
(B) Ping–pong mechanisms (appendix 4B) 13
6. The metal ion 13
A. Zinc 13
B. Magnesium 14
C. Copper 14
D. Manganese 15
E. Cobalt 15
F. Nickel 15
G. Calcium 15
7. Inhibitor types for enzyme activities 16
Types of reversible inhibitors 16
8. Specific aimed 18
Materials and Methods 19
1. Expression of native rec-HP0642 protein 19
2. Purification of native rec-HP0642 protein 19
3. Function assays for rec-HP0642 19
4. Nitroreductase assay 20
A. The nitrofurazone reduction 20
B. The nitrofurantoin reduction 20
5. Kinetic Studies 21
6. Inhibitor Studies 22
7. Divalent metal ions 22
Results 23
I. Oxidoreductase activity of recHP0642 23
A. NADH:flavin Oxidoreductase activity of recHP0642 protein 23
B. The effect of divalent metal ion on NADH oxidation activity of recHP0642 protein 23
II. Nitroreductase activity of recHP0642 25
A. Nitrofurazone nitroreductase activity of recHP0642 25
B. Nitrofurantoin nitroreductase activity of recHP0642 26
Discussion 28
1. Choices for divalent metal ions 28
2. NADH or NADPH preference in this study 28
3. Effects of metal ions on NADH:flavin oxidoreductase activity 29
4. The effects of metal ions on nitrofurazone reduction activity of recHP0642 30
5. The effects of metal ions on nitrofurantoin reduction activity of recHP0642 30
6. The Cu2+ inhibition type for nitroreductase activity of recHP0642 preotein 31
References 32
Figures 38
Figure 1. NADH oxidoreductase activity assay of recHP0642 39
Figure 2. Comparison of NADH oxidase activities of recHP0642 in the presence of metal ions 42
Figure 3. Nitrofuranzone nitroreductase activities of recHP0642 44
Figure 4. The effect of divalent metal ion on nitrofuranzone reduction activity of recHP0642 45
Figure 5. The effects of 0.2 mM Cu2+ on rec-HP0642 nitrofurazone nitroreductase activity 47
Figure 6. The Cu2+ inhibition type of recHP0642 nitrofurazone nitroreduction activity 48
Figure 7. Nitrofurantoin nitroreductase for recHP0642 protein 50
Figure 8. The effect of divalent metal ion on nitrofurantoin reduction activity of recHP0642 51
Figure 9. The effect of Cu2+ at 0.02 mM on nitrofurantoin reduction activity of recHP0642 protein 52
Figure 10. The effects of 0.02 mM Cu2+ on rec-HP0642 nitrofurantoin nitroreductase activity 54
Figure 11. The Cu2+ inhibition type of recHP0642 nitrofurantoin nitroreduction activity 55
Tables 56
Table 1. Comparison of NADH oxidase activity of recHP0642 in the presence of 2 mM metal ions 56
Table 2. Comparison of NADH oxidase activity of recHP0642 in the presence of 16 mM metal ions 56
Appendix 57
1. Michaelis-Menten saturation curve of an enzyme reaction 57
2. Mechanism plus rates 57
3. Lineweaver-Burke plot 57
4. Multi-substrate reactions 58
5. Nitro compounds 58
6. NADH is abbreviated from Nicotinamide adenine dinucleotide. 59
7. Mechanism for reducing nitrofurazone with FMN 59
8. FMN is abbreviated from Flavin mononucleotide. 60
9. Some reactions about flavin reductase 60


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