The evolutionary conservation of the non-covalent interactions in the Class A β-lactamase family was analyzed using Key Interaction Networks (KIN). This analysis built on prior work considering the evolution of interaction networks in these enzymes, now including also the GNCA4-12 variant in the analysis. As in our prior work, 60,61 TEM-1 was selected the reference structure for projection of the network, owing both to its evolutionary connection to the chosen ancestral sequences, and its chemical significance as a catalytic specialist. The KIN calculated conservation network provides a relative conservation score for each interacting residue pair, based on how frequently each interaction appears across the structures. A cutoff of 50% conservation was used in interaction analysis. From this, per residue KIN evolutionary conservation scores were calculated for direct comparison to the KIF analysis.