The Role of a Second-Shell Residue in Modifying Substrate and Inhibitor Interactions in the SHV β-Lactamase: A Study of Ambler Position Asn276

Inhibitor-resistant class A beta-lactamases of the TEM and SHV families that arise by single amino acid substitutions are a significant threat to the efficacy of beta-lactam/beta-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 colt, only the Asn276Asp enzyme demonstrated reduced Susceptibility to ampicillin/clavulanate (MIC increased from 50/2 -> 50/8 mu g/mL) while maintaining high-level resistance to ampicillin (MIC = 8 192 mu g/mL). steady-state kinetic analyses of Asn276Asp revealed slightly diminished k(cat)/K-m for all Substrates tested. In contrast, we observed a 5-fold increase in K-i for clavulanate (7.4 +/- 0.9 mu M for Asn276Asp vs 1.4 +/- 0.2 mu M for SHV-1) and a 40% reduction in k(inact)/K-1 (0.013 +/- 0.002 mu M-1 s(-1) for Asn276Asp vs 0.021 +/- 0.004 mu M-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 Arg244N eta 2 and both 276AspO delta 1 and O delta 2; these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C-3/C-4 carboxylate of beta-lactam substrates and inhibitors. Testing the functional consequences of this interaction, we noted considerable free energy costs (+Delta Delta G) for substrates and inhibitors. A rigid carbapenem (meropenem) was most affected by the Asn276Asp substitution (46-fold increase in K-i vs SHV-1). We conclude that residue 276 is an important second-shell residue in class A beta-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.