The rise of multi-drug resistance in bacterial pathogens is one of the grand challenges facing medical science. A major concern is the speed of development of β-lactamase-mediated resistance in Gram-negative species, thus putting at risk the efficacy of the most recently approved antibiotics and inhibitors, including carbapenems and avibactam, respectively. New strategies to overcome resistance are urgently required, which will ultimately be facilitated by a deeper understanding of the mechanisms that regulate the function of β-lactamases such as the Klebsiella Pneumoniae carbapenemases (KPCs). Using enhanced sampling computational methods together with site-directed mutagenesis, we report the identification of two “hydrophobic networks” in the KPC-2 enzyme, the integrity of which has been found to be essential for protein stability and corresponding resistance. Present throughout the structure, these networks are responsible for the structural integrity and allosteric signaling. Disruption of the networks leads to a loss of the KPC-2 mediated resistance phenotype, resulting in restored susceptibility to different classes of β-lactam antibiotics including carbapenems and cephalosporins. The ”hydrophobic networks” were found to be highly conserved among class-A β-lactamases, which implies their suitability for exploitation as a potential target for therapeutic intervention.