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

Abstract

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with ␤-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize ␤-lactam antibiotics, it is important to know their energies of interaction. Because ␤-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between ␤-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 ␤-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 (k 2 /KЈ). 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; ␤-lactam; ␤-lactamase; enzyme stability; denaturation Penicillin-binding proteins (PBPs) are involved in the biosynthesis of bacterial cell wall peptidoglycan. All bacteria analyzed to date have multiple PBPs, which function to crosslink the cell wall peptides (transpeptidation) and to remove the C terminal D-alanine residue from cell wall peptides (carboxypeptidation). PBPs are the targets of ␤-lactam antibiotics, which react with these enzymes to form stable covalent complexes, leading to inactivation of the PBP and ultimately to cell death (Tipper and Strominger 1965;Wise and Park 1965;Ghuysen and Dive 1994). A broad range of ␤-lactam antibiotics, including penicillins, cephalosporins, monobactams, and carbapenems, are used clinically, and seemingly subtle structural differences have dramatic effects on their biological activity (Neuhaus and Georgopapadakou 1992).Despite much interest in understanding the molecular interactions of ␤-lactams with PBPs, their interaction energies have been difficult to determine. The noncovalent first encounter complex between the antibiotic and enzyme proceeds quickly to an acyl-enzyme adduct by attack of the

The Role of the β5–α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

“…Our findings are similar, however, to those we reported for E. coli PBP 5, where, in crystal structures of acylated complexes, electron density for the R1/R2 substituents of various β-lactams was also consistently weak and did not correlate with acylation rates 29 . Such data reinforce the notion that acylated complexes of PBPs with β-lactams may only be of limited value when rationally designing new inhibitors of PBPs because these may not reveal interactions that occurred during acylation 23 . In support of this, crystal structures of the AmpC β-lactamase in the pre-covalent, acylated and post-covalent product state show significantly different modes of binding for cephalothin 30 .…”

“…For N. gonorrhoeae PBP 2, two cephalosporins (ceftriaxone and cefixime) both exhibit markedly higher rates of acylation compared to penicillin G 18 , which is opposite to PBPA. Finally, for E. coli PBP 5, the corresponding order of acylation rates is cefoxitin (a cephalosporin) > imipenem > cloxacillin (a penicillin) 23 . Two points emerge from such data: 1) the acylation rates vary for the same antibiotic against different PBPs, and 2) rates for different antibiotics directed against the same PBP also vary and appear specific for that target.…”

Kinetics of β-Lactam Interactions with Penicillin-susceptible and -resistant Penicillin-binding Protein 2x Proteins from Streptococcus pneumoniae

“…low K d values, appears not to be required for achieving high potency. Consistent with these conclusions is the recent observation that k 2 /K d values of a PBP with different ␤-lactams do not correlate with the interaction energies (which reflect the complementarity of a ligand) between the PBP and the ␤-lactams based on thermal denaturation studies of E. coli PBP5, indicating that a complementary fit of a ␤-lactam to PBP is not required for acylation (33).…”

Crystal Structures of Covalent Complexes of β-Lactam Antibiotics with Escherichia coli Penicillin-Binding Protein 5: Toward an Understanding of Antibiotic Specificity

“…Excluding atoms that were not visible in the electron density ( i.e. were modeled with zero occupancy), the area occluded by antibiotic in each of the three complexes was calculated and compared with the second order rate constant of acylation and the non-covalent interaction energy for the same antibiotics, as determined by Beadle et al (12) (Table 2). Even though cloxacillin occludes the greatest surface area of PBP 5 (258 Å 2 ), it exhibits the lowest rate of acylation with PBP 5.…”

“…Whilst it might be assumed that, like non-covalent inhibitors, β-lactams with the highest affinity and therefore with the highest complementarity with the active site of a PBP would be the most potent inhibitors, PBPs do not bind β-lactams more tightly than peptide substrates (13), making it unlikely that the non-covalent affinity is the primary parameter that dictates the efficacy of enzyme inactivation. In support of this, there is a lack of correlation between the non-covalent interaction energy (a measure of complementarity) of a β-lactam complex of E. coli PBP 5 and the k 2 / K S value of that β-lactam against the same PBP (12). A related issue is whether induced fit contributes to the inhibition of PBPs by β-lactams because the highest extent of interaction might occur in the acylation transition state.…”