Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site

beta -Lactamases hydrolyze beta -lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechan ism to these drugs and pose a growing threat to public health. beta -Lactam s that contain a bulky 6(7)a substituent, such as imipenem and moxalactam, actually inhibit serine beta -lactamases and are widely used for this reaso n. Although mutant serine p-lactamases have arisen that hydrolyze beta -lac tamase resistant beta -lactams (e,g,, ceftazidime) or avoid mechanism-based inhibitors (e.g,, clavulanate), mutant serine beta -lactamases have not ye t arisen in the clinic with imipenemase or moxalactamase activity, Structur al and thermodynamic studies suggest that the 6(7)a substituents of these i nhibitors form destabilizing contacts within the covalent adduct with the c onserved Asn152 in class C beta -lactamases (Asn132 in class A beta -lactam ases), This unfavorable interaction may be crucial to inhibition. To test t his destabilization hypothesis, we replaced Asn152 with Ala in the class C beta -lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consisten t with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1. 10 kcal/mol of strain introduced by imipenem and moxalactam. respectively, relative to the wild-type complexes. However, the kinetic efficiency of Amp C N152A was reduced by 6300-fold relative to that of the wild-type enzyme, To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determi ned to a resolution of 1.83 Angstrom. Moxalactam in the mutant complex is s ignificantly displaced from its orientation in the wild-type complex; howev er, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta -lactams with 6(7)alpha substit uents out of a catalytically competent configuration, making them inhibitor s, the residue is essential for orienting beta -lactam substrates and canno t simply be replaced with a much smaller residue to restore catalytic activ ity. Designing beta -lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.