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FOR THE RECORD

Interaction energies between ß-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation

¹ Department of Molecular Pharmacology & Biological Chemistry, Northwestern University, Chicago, Illinois 60611-3008, USA
² Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365, USA

Reprint requests to: Brian K. Shoichet, Department of Molecular Pharmacology & Biological Chemistry, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611-3008, USA; e-mail: b-shoichet{at} northwestern.edu; fax: (312) 503-5349.

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with ß-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize ß-lactam antibiotics, it is important to know their energies of interaction. Because ß-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between ß-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme–antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T_m) of 48.5°C and a van't Hoff enthalpy of unfolding (H_VH) of 193 kcal/mole. The binding of the ß-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T_m values as high as +4.6°C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k₂/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.

Keywords: Penicillin-binding protein; PBP 5; ß-lactam; ß-lactamase; enzyme stability; denaturation

Abbreviations: [¹²⁵I]IPV, [¹²⁵I]penicillin V • C_p, change in heat capacity at constant pressure • G_interaction, noncovalent interaction energy • G_u, Gibbs free energy of unfolding • H_VH, van't Hoff enthalpy of unfolding • S_u, entropy of unfolding • k₂/K', second-order rate constant • K_eq, equilibrium constant • PBP, penicillin-binding protein • T_m, temperature of melting

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