Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability
Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability
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DOI：
10.1021/bi971056h
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发表时间：
1997-09-23
期刊：
影响因子：
2.9
通讯作者：
Galleni, M
中科院分区：
文献类型：
--
作者：
Valladares, MH;Felici, A;Galleni, M
在嗜水气单胞菌AE 036金属β-内酰胺酶中发现了两个Zn ~（2+）结合位点。第一个结合位点对Zn 2+离子的亲和力很高，以至于不能确定解离常数，但它显著低于20 nM。该酶的单-Zn 2+形式对其碳青霉烯底物表现出最大活性。在第二较低亲和力结合位点中存在Zn 2+离子导致酶活性的损失，在pH 6.5时Ki值为46 μ M。动力学分析符合非竞争性抑制机理。结果表明：A. Escherichia酶也具有强烈的pH依赖性。与外部的Zn 2+离子浓度为0.4 μ M，由金属离子的高亲和力位点的占用率低于10%，在pH 5和10。对第二结合位点的亲和力似乎从pH 6增加到7.5。荧光发射光谱和圆二色光谱显示轻微的构象变化后，由Zn 2+离子滴定的脱辅基酶，导致在连续饱和的第一和第二个结合位点。差示扫描量热法转换和内在荧光发射光谱中存在的尿素浓度的增加表明，催化锌强烈稳定的构象的酶，而二锌酶是更耐热和尿素变性比单锌酶。因此，这种金属-β-内酰胺酶的活性的Zn 2+依赖性似乎与同源脆弱拟杆菌酶的活性非常不同，对于所述同源脆弱拟杆菌酶，每分子蛋白质存在两个Zn 2+离子似乎导致最大活性。
Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a K-i value of 46 mu M at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 mu M, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.