Inhibitor-resistant class A β-lactamases of the TEM and SHV families that arise by single amino acid substitutions are a significant threat to the efficacy of β-lactam/β-lactamase inhibitor combinations. To better understand the basis of the inhibitor-resistant phenotype in SHV, we performed mutagenesis to examine the role of a second-shell residue, Asn276. Of the 19 variants expressed in Escherichia coli, only the Asn276Asp enzyme demonstrated reduced susceptibility to ampicillin/clavulanate (MIC increased from 50/2 → 50/8 μg/mL) while maintaining high-level resistance to ampicillin (MIC = 8192 μg/mL). Steady-state kinetic analyses of Asn276Asp revealed slightly diminished kcat/Km for all substrates tested. In contrast, we observed a 5-fold increase in Ki for clavulanate (7.4 ± 0.9 μM for Asn276Asp vs 1.4 ± 0.2 μM for SHV-1) and a 40% reduction in kinact/KI (0.013 ± 0.002 μM-1 s-1 for Asn276Asp vs 0.021 ± 0.004 μM-1 s -1 for SHV-1). Timed electrospray ionization mass spectrometry of clavulanate-inhibited SHV-1 and SHV Asn276Asp showed nearly identical mass adducts, arguing for a similar pathway of inactivation. Molecular modeling shows that novel electrostatic interactions are formed between Arg244Nη2 and both 276AspOδ1 and Oδ2; these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C3/C 4 carboxylate of β-lactam substrates and inhibitors. Testing the functional consequences of this interaction, we noted considerable free energy costs ( + ΔΔG) for substrates and inhibitors. A rigid carbapenem (meropenem) was most affected by the Asn276Asp substitution (46-fold increase in Ki vs SHV-1). We conclude that residue 276 is an important second-shell residue in class A β-lactamase-mediated resistance to substrates and inhibitors, and only Asn is able to precisely modulate the conformational flexibility of Arg244 required for successful evolution in nature.