Departamento de Química Biológica and Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina.
J Biol Chem. 2010 Feb 12;285(7):4570-7. doi: 10.1074/jbc.M109.063743. Epub 2009 Dec 10.
Metallo-beta-lactamases (MbetaLs) stand as one of the main mechanisms of bacterial resistance toward carbapenems. The rational design of an inhibitor for MbetaLs has been limited by an incomplete knowledge of their catalytic mechanism and by the structural diversity of their active sites. Here we show that the MbetaL GOB from Elizabethkingia meningoseptica is active as a monometallic enzyme by using different divalent transition metal ions as surrogates of the native Zn(II) ion. Of the metal derivatives in which Zn(II) is replaced, Co(II) and Cd(II) give rise to the most active enzymes and are shown to occupy the same binding site as the native ion. However, Zn(II) is the only metal ion capable of stabilizing an anionic intermediate that accumulates during nitrocefin hydrolysis, in which the C-N bond has already been cleaved. This finding demonstrates that the catalytic role of the metal ion in GOB is to stabilize the formation of this intermediate prior to nitrogen protonation. This role may be general to all MbetaLs, whereas nucleophile activation by a Zn(II) ion is not a conserved mechanistic feature.
金属β-内酰胺酶（MbetaLs）是细菌对碳青霉烯类抗生素产生耐药性的主要机制之一。由于对其催化机制了解不完整，以及其活性位点结构的多样性，针对 MbetaLs 的抑制剂的合理设计受到了限制。在这里，我们通过使用不同的二价过渡金属离子作为天然 Zn(II)离子的替代品，表明脑膜脓毒性伊丽莎白菌的 MbetaL GOB 作为单金属酶发挥作用。在取代 Zn(II)的金属衍生物中，Co(II)和 Cd(II)产生最活跃的酶，并被证明占据与天然离子相同的结合位点。然而，只有 Zn(II)金属离子能够稳定在 nitrocefin 水解过程中积累的阴离子中间体，其中 C-N 键已经被切断。这一发现表明，金属离子在 GOB 中的催化作用是在氮质子化之前稳定这种中间体的形成。这一作用可能对所有 MbetaLs 都是通用的，而 Zn(II)离子对亲核试剂的激活不是保守的机制特征。
