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).

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