Functional analyses of AmpC beta-lactamase through differential stability.

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Beta-lactam antibiotics are crucial, but their binding to target enzymes like beta-lactamases is not fully understood.
• The rapid covalent intermediate formation during beta-lactam binding hinders direct measurement of interaction energies.

Purpose of the Study:

• To explore the energetic complementarity of beta-lactams with the beta-lactamase AmpC using reversible denaturation.
• To investigate the role of specific residues in AmpC's interaction with beta-lactams.

Main Methods:

• Reversible thermal and solvent denaturation of AmpC and its adducts with various beta-lactams.
• Measurement of changes in enzyme stability (Tm, deltaG) upon ligand binding.
• Analysis of a catalytically inactive AmpC mutant (Y150F).

Main Results:

• AmpC denaturation followed a two-state model.
• Penicillin cloxacillin and monobactam aztreonam stabilized AmpC, indicating favorable interactions.
• Oxacephem moxalactam and carbapenem imipenem destabilized AmpC, suggesting unfavorable noncovalent interactions.
• Transition state analogs and the Y150F mutant also showed altered stability profiles.

Conclusions:

• Reversible denaturation provides a method to energetically analyze beta-lactam complementarity with AmpC.
• Ligand binding significantly perturbs enzyme stability, with varying effects based on drug structure.
• Specific residues, like Y150, may be critical for recognizing enzyme-ligand interactions.