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

Abstract

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).