Llarrull Leticia I, Tioni Mariana F, Vila Alejandro J
Instituto de Biologia Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Cientificas y Tecnicas de Argentina, S2002LRK Rosario, Argentina.
J Am Chem Soc. 2008 Nov 26;130(47):15842-51. doi: 10.1021/ja801168r.
Metallo-beta-lactamases are enzymes capable of hydrolyzing all known classes of beta-lactam antibiotics, rendering them ineffective. The design of inhibitors active against all classes of metallo-beta-lactamases has been hampered by the heterogeneity in metal content in the active site and the existence of two different mononuclear forms. BcII is a B1 metallo-beta-lactamase which is found in both mononuclear and dinuclear forms. Despite very elegant studies, there is still controversy on the nature of the active BcII species. We carried out a non-steady-state study of the hydrolysis of penicillin G catalyzed by Co(II)-substituted BcII, and we followed the modifications occurring at the active site of the enzyme. Working at different metal/enzyme ratios we demonstrate that both mono-Co(II) and di-Co(II) BcII are active metallo-beta-lactamases. Besides, we here present evidence that during penicillin G hydrolysis catalyzed by mono-Co(II) BcII the metal is localized in the DCH site (the Zn2 site in B1 enzymes). These conclusions allow us to propose that both in mono-Co(II) and di-Co(II) BcII the substrate is bound to the enzyme through interactions with the Co(II) ion localized in the DCH site. The finding that the DCH site is able to give rise to an active lactamase suggests that the Zn2 site is a common feature to all subclasses of metallo-beta-lactamases and would play a similar role. This proposal provides a starting point for the design of inhibitors based on transition-state analogs, which might be effective against all MbetaLs.
金属β-内酰胺酶是一类能够水解所有已知类型β-内酰胺抗生素，使其失效的酶。针对所有类型金属β-内酰胺酶的抑制剂设计，因活性位点金属含量的异质性以及两种不同单核形式的存在而受到阻碍。BcII是一种B1型金属β-内酰胺酶，存在单核和双核两种形式。尽管有非常出色的研究，但关于活性BcII物种的性质仍存在争议。我们对钴（II）取代的BcII催化青霉素G水解进行了非稳态研究，并追踪了酶活性位点发生的变化。在不同的金属/酶比例下进行研究，我们证明单钴（II）和双钴（II）BcII都是有活性的金属β-内酰胺酶。此外，我们在此提供证据表明，在单钴（II）BcII催化青霉素G水解过程中，金属定位于DCH位点（B1型酶中的Zn2位点）。这些结论使我们能够提出，在单钴（II）和双钴（II）BcII中，底物都是通过与定位于DCH位点的钴（II）离子相互作用而与酶结合的。DCH位点能够产生有活性的内酰胺酶这一发现表明，Zn2位点是所有金属β-内酰胺酶亚类的共同特征，并且可能发挥类似的作用。这一观点为基于过渡态类似物设计抑制剂提供了一个起点，这类抑制剂可能对所有金属β-内酰胺酶都有效。
