Abstract
We report an extensive survey of the inhibition effect of graphene oxide (GO) on the enzymatic activity of β-lactamases (BLs), namely: serine β-lactamases (SBLs) SHV-1, CTX-M-15, GES-1, KPC-3, OXA-23, and metallo-β-lactamases (MBLs) (NDM-1, VIM-1, CphA, and L1). The inhibition study has been performed with GO dose-dependent kinetic assays (using nitrocefin as a substrate). On all the SBLs, a formidable inhibition activity is demonstrated already at a GO concentration of 0.5 µg ml(-1). Inhibition is observed for all the MBLs in the monomeric phase. For MBLs, the interaction mechanism with GO has been directly investigated with X-ray photoemission spectroscopy, focusing on the interaction of NDM-1 with GO. Data do not show any specific interaction with GO of the Zn atoms at the active site of the enzyme, while a detailed analysis of the C 1s and N 1s core levels points to the interaction of GO with the enzyme via its positive residues, indicating a non-competitive inhibition mechanism. Together with the modeling of the nitrocefin-GO system with density functional theory, we demonstrate that the GO basal plane links the substrate molecule via oxidation of one of the two sulfur atoms of the substrate, strongly reducing the inhibition effectiveness if GO is pre-incubated with the substrate.