Hernandez Valladares M, Felici A, Weber G, Adolph H W, Zeppezauer M, Rossolini G M, Amicosante G, Frère J M, Galleni M
Centre d'Ingénierie des Protéines (B6) and IPNE (B15), Université de Liège, Sart Tilman, Liège 4000, Belgium.
Biochemistry. 1997 Sep 23;36(38):11534-41. doi: 10.1021/bi971056h.
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 Ki value of 46 microM 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 microM, 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.
在嗜水气单胞菌AE036金属β-内酰胺酶中发现了两个锌离子结合位点。第一个结合位点对锌离子的亲和力极高，以至于无法测定其解离常数，但明显低于20 nM。该酶的单锌形式对其碳青霉烯底物表现出最大活性。第二个低亲和力结合位点中存在锌离子会导致酶活性丧失，在pH 6.5时的Ki值为46 μM。动力学分析与非竞争性抑制机制一致。嗜水气单胞菌酶的锌含量也强烈依赖于pH值。在外部锌离子浓度为0.4 μM时，在pH 5和10条件下，高亲和力位点被金属离子占据的比例低于10%。对第二个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，在脱辅基酶用锌离子滴定过程中发生了轻微的构象变化，导致第一个和第二个结合位点相继饱和。差示扫描量热法转变以及在尿素浓度增加时的固有荧光发射光谱表明，催化锌强烈稳定酶的构象，而双锌酶比单锌酶对热变性和尿素变性更具抗性。因此，这种金属β-内酰胺酶活性对锌离子的依赖性似乎与脆弱拟杆菌同源酶有很大不同，对于后者，每分子蛋白质存在两个锌离子似乎会导致最大活性。
