Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation.
Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-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 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees 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 (k(2)/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.
Beadle
B M
BM
Department of Molecular Pharmacology & Biological Chemistry, Northwestern University, Chicago, Illinois 60611-3008, USA.
Nicholas
R A
RA
Shoichet
B K
BK
D000215:Acylation; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002440:Cefoxitin / Q000378:metabolism; D002942:Circular Dichroism; D003023:Cloxacillin / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D006602:Hexosyltransferases; D015378:Imipenem / Q000378:metabolism; D008956:Models, Chemical; D009070:Moxalactam / Q000378:metabolism; D002267:Muramoylpentapeptide Carboxypeptidase / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011485:Protein Binding; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D047090:beta-Lactams / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D047090:beta-Lactams; D002440:Cefoxitin; D015378:Imipenem; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D003023:Cloxacillin; D009070:Moxalactam