Enzyme graphene oxide interaction: the case system of β-lactamases

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 <i>via</i> 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 <i>via</i> oxidation of one of the two sulfur atoms of the substrate, strongly reducing the inhibition effectiveness if GO is pre-incubated with the substrate.