β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k(cat)/K(m)). In addition, we find that residue 220 is central to the mechanism of β-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively "inhibitor susceptible" (K(i) of 14 ± 1 μM for clavulanic acid versus K(i) of 25 ± 2 μM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K(m) values for β-lactamase inhibitors (>100 μM for clavulanic acid versus 25 ± 3 μM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A β-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the "relatively" clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.