| Abstract
| - β-Lactamases are the most widespread resistance mechanism to β-lactam antibiotics, such asthe penicillins and cephalosporins. Transition-state analogues that bind to the enzymes with nanomolaraffinities have been introduced in an effort to reverse the resistance conferred by these enzymes. Tounderstand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamiccycle experiments were undertaken. An unexpected hydrogen bond between the nonconserved Asn289and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nM inhibitor (compound1) in complex with AmpC β-lactamase. To investigate the energy of this hydrogen bond, the mutantenzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2). Thedifferential affinity of the four different protein and analogue complexes indicates that the carboxylate−amide hydrogen bond contributes 1.7 kcal/mol to overall binding affinity. Synthesis of an analogue of 1where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bondenergy, consistent with the importance of the ionic nature of this hydrogen bond. To investigate the structuralbases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and1.39 Å, respectively. These structures suggest that no significant rearrangement occurs in the mutantversus the wild-type complexes with both compounds. The mutant enzymes L119A and L293A weremade to investigate the interaction between a phenyl ring in 1 and these residues. Whereas deletion of thephenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does notcome through interaction with the leucines, despite the close contact in the structure. The energies ofthese interactions provide key information for the design of improved inhibitors against β-lactamases.The high magnitude of the ion−dipole interaction between Asn289 and the carboxylate of 1 is consistentwith the idea that ionic interactions can provide significant net affinity in inhibitor complexes.
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