Objectives: Investigation into the adenylylation of the nucleophilic serine in AmpC BER and CMY-10 extended-spectrum class C β-lactamases. Methods: The formation and the stability of the adenylate adduct were examined by X-ray crystallography and MS. Inhibition assays for kinetic parameters were performed by monitoring the hydrolytic activity of AmpC BER and CMY-10 using nitrocefin as a reporter substrate. The effect of adenosine 5′-(P-acetyl)monophosphate (acAMP) on the MIC of ceftazidime was tested with four Gram-negative clinical isolates. Results: The crystal structures and MS analyses confirmed the acAMP-mediated adenylylation of the nucleophilic serine in AmpC BER and CMY-10. acAMP inhibited AmpC BER and CMY-10 through the adenylylation of the nucleophilic serine, which could be modelled as a two-step mechanism. The initial non-covalent binding of acAMP to the active site is followed by the covalent attachment of its AMP moiety to the nucleophilic serine. The inhibition efficiencies (kinact/KI) of acAMP against AmpC BER and CMY-10 were determined to be 320 and 140 M-1 s-1, respectively. The combination of ceftazidime and acAMP reduced the MIC of ceftazidime against the tested bacteria. Conclusions: Our structural and kinetic studies revealed the detailed mechanism of adenylylation of the nucleophilic serine and may serve as a starting point for the design of novel class C β-lactamase inhibitors on the basis of the nucleotide scaffold.