Noncovalent interaction energies in covalent complexes: Tem-1 β-lactamase and β-lactams

Xiaojun Wang, George Minasov, Brian K. Shoichet*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

40 Scopus citations


The class A β-lactamase TEM-1 is a key bacterial resistance enzyme against β-lactam antibiotics, but little is known about the energetic bases for complementarity between TEM-1 and its inhibitors. Most inhibitors form a covalent adduct with the catalytic Ser70, making the measurement of equilibrium constants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions within covalent complexes by examining the differential stability of TEM-1 and its inhibitor adducts. The thermal denaturation of TEM-1 follows a two-state, reversible model with a melting temperature (Tm) of 51.6°C and a van't Hoff enthalpy of unfolding (ΔHvH) of 146.2 kcal/mol at pH 7.0. The stability of the enzyme changes on forming an inhibitor adduct. As expected, some inhibitors stabilize TEM-1; transition-state analogues increase the Tm by up to 3.7°C (1.7 kcal/mol). Surprisingly, all β-lactam covalent acyl-enzyme complexes tested destabilize TEM-1 significantly relative to the apoenzyme. For instance, the clinically used inhibitor clavulanic acid and the β-lactamase-resistant β-lactams moxalactam and imipenem destabilize TEM-1 by over 2.6°C (1.2 kcal/mol) in their covalent adducts. Based on the structure of the TEM-1/imipenem complex (Maveyraud et al., J Am Chem Soc 1998;120-9748-52), destabilization by moxalactam and imipenem is thought to be caused by a steric clash between the side-chain of Asn132 and the 6(7)-α group of these β-lactams. To test this hypothesis, the mutant enzyme N132A was made. In contrast with wild-type, the covalent complexes between N132A and both imipenem and moxalactam stabilize the enzyme, consistent with the hypothesis. To investigate the structural bases of this dramatic change in stability, the structure of N132A/ imipenem was determined by X-ray crystallography. In the complex with N132A, imipenem adopts a very different conformation from that observed in the wild-type complex, and the putative destabilizing interaction with residue 132 is relieved. Studies of several enzymes suggest that β-lactams, and covalent inhibitors in general, can have either net favorable or net unfavorable noncovalent interaction energies within the covalent complex. In the case of TEM-1, such unfavorable interactions convert substrate analogues into very effective inhibitors.

Original languageEnglish (US)
Pages (from-to)86-96
Number of pages11
JournalProteins: Structure, Function and Genetics
Issue number1
StatePublished - Apr 1 2002


  • Antibiotic resistance
  • Protein stability
  • TEM-1
  • β-lactamase

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology


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