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Title: Noncovalent Interaction Energies in Covalent Complexes: TEM-1 beta-Lactamase and beta-Lactams

Abstract

The class A {beta}-lactamase TEM-1 is a key bacterial resistance enzyme against {beta}-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 equilibriumconstants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions withincovalent 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 (T{sub m}) of 51.6 C and a van't Hoff enthalpy of unfolding ({Delta}H{sub VH}) 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 T{sub m} by up to 3.7 C(1.7 kcal/mol). Surprisingly, all {beta}-lactam covalent acyl-enzyme complexes tested destabilize TEM-1 significantly relative to the apoenzyme. For instance, the clinically used inhibitor clavulanic acid and the {beta}-lactamase-resistant {beta}-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 amore » steric clash between the side-chain of Asn132 and the 6(7)-{alpha} group of these {beta}-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 instability, the structure of N132A/imipenem was determined by X-ray crystallography. In the complex with N132A, imipenemadopts 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 {beta}-lactams, andcovalent 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.« less

Authors:
; ;  [1]
  1. NWU
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1008801
Resource Type:
Journal Article
Journal Name:
Proteins
Additional Journal Information:
Journal Volume: 47; Journal Issue: 2002; Journal ID: ISSN 0887-3585
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; ADDUCTS; ANTIBIOTICS; CRYSTALLOGRAPHY; ENTHALPY; ENZYMES; HYPOTHESIS; INSTABILITY; MELTING; MUTANTS; RESIDUES; STABILITY; SUBSTRATES

Citation Formats

Wang, Xiaojun, Minasov, George, and Shoichet, Brian K. Noncovalent Interaction Energies in Covalent Complexes: TEM-1 beta-Lactamase and beta-Lactams. United States: N. p., 2010. Web.
Wang, Xiaojun, Minasov, George, & Shoichet, Brian K. Noncovalent Interaction Energies in Covalent Complexes: TEM-1 beta-Lactamase and beta-Lactams. United States.
Wang, Xiaojun, Minasov, George, and Shoichet, Brian K. 2010. "Noncovalent Interaction Energies in Covalent Complexes: TEM-1 beta-Lactamase and beta-Lactams". United States.
@article{osti_1008801,
title = {Noncovalent Interaction Energies in Covalent Complexes: TEM-1 beta-Lactamase and beta-Lactams},
author = {Wang, Xiaojun and Minasov, George and Shoichet, Brian K},
abstractNote = {The class A {beta}-lactamase TEM-1 is a key bacterial resistance enzyme against {beta}-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 equilibriumconstants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions withincovalent 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 (T{sub m}) of 51.6 C and a van't Hoff enthalpy of unfolding ({Delta}H{sub VH}) 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 T{sub m} by up to 3.7 C(1.7 kcal/mol). Surprisingly, all {beta}-lactam covalent acyl-enzyme complexes tested destabilize TEM-1 significantly relative to the apoenzyme. For instance, the clinically used inhibitor clavulanic acid and the {beta}-lactamase-resistant {beta}-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)-{alpha} group of these {beta}-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 instability, the structure of N132A/imipenem was determined by X-ray crystallography. In the complex with N132A, imipenemadopts 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 {beta}-lactams, andcovalent 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.},
doi = {},
url = {https://www.osti.gov/biblio/1008801}, journal = {Proteins},
issn = {0887-3585},
number = 2002,
volume = 47,
place = {United States},
year = {Mon Mar 08 00:00:00 EST 2010},
month = {Mon Mar 08 00:00:00 EST 2010}
}