Mechanism of turnover of imipenem by the TEM .beta.-lactamase revisited

Taibi, Pascale; Mobashery, Shahriar

doi:10.1021/ja00134a003

Cited by 57 publications

(72 citation statements)

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“…The rate at which imipenem was displaced from the active site of GES-1, -2, and -5 was determined as described previously (21). Briefly, a reaction containing 50 mM NaP i (pH 7.0), 100 mM NaCl, and 1 M GES-1 and -2 or 750 nM GES-5 in the absence and presence of 20 M (in the case of GES-1), 5 M (in the case of GES-2), or 15 M (in the case of GES-5) imipenem (10 ϫ K m ) was incubated at room temperature for 2 min.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of β-Lactamases

Frase

Shi

Testero

et al. 2009

Journal of Biological Chemistry

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A major mechanism of bacterial resistance to ␤-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is the production of ␤-lactamases. A handful of class A ␤-lactamases have been discovered that have acquired the ability to turn over carbapenem antibiotics. This is a disconcerting development, as carbapenems are often considered last resort antibiotics in the treatment of difficult infections. The GES family of ␤-lactamases constitutes a group of extended spectrum resistance enzymes that hydrolyze penicillins and cephalosporins avidly. A single amino acid substitution at position 170 has expanded the breadth of activity to include carbapenems. The basis for this expansion of activity is investigated in this first report of detailed steady-state and pre-steady-state kinetics of carbapenem hydrolysis, performed with a class A carbapenemase. Monitoring the turnover of imipenem (a carbapenem) by GES-1 (Gly-170) revealed the acylation step as rate-limiting. GES-2 (Asn-170) has an enhanced rate of acylation, compared with GES-1, and no longer has a single rate-determining step. Both the acylation and deacylation steps are of equal magnitude. GES-5 (Ser-170) exhibits an enhancement of the rate constant for acylation by a remarkable 5000-fold, whereby the enzyme acylation event is no longer rate-limiting. This carbapenemase exhibits k cat /K m of 3 ؋ 10 5 M ؊1 s ؊1 , which is sufficient for manifestation of resistance against imipenem.

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Section: Methodsmentioning

confidence: 99%

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of β-Lactamases

Frase

Shi

Testero

et al. 2009

Journal of Biological Chemistry

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“…Further work by Mobashery's group demonstrated that acylation of TEM-1 by imipenem occurs readily and is followed by ⌬ 2 -to ⌬ 1 -tautomerization of the pyrroline double bond in the acyl-enzyme species (Fig. 6) (407). The rate of deacylation differs for the tautomers (the ⌬ 1 tautomer is approximately 10-fold slower than the ⌬ 2 tautomer), and it was initially accepted that molecular rearrangements to the more stable (i.e., more slowly deacylated) species accounted for enzyme inhibition.…”

Section: Inhibitory Activity Of Carbapenemsmentioning

confidence: 99%

“…The rate of deacylation differs for the tautomers (the ⌬ 1 tautomer is approximately 10-fold slower than the ⌬ 2 tautomer), and it was initially accepted that molecular rearrangements to the more stable (i.e., more slowly deacylated) species accounted for enzyme inhibition. Site-directed mutagenesis studies of TEM-1 Arg244Ser showed that the guanidinium group on Arg244 provides directly, or indirectly via a water molecule, the necessary proton for tautomerization to the ⌬ 1 -pyrroline acyl-enzyme (407,458). Incidentally, this water appears to be the same molecule that serves as the proton donor necessary for clavulanate inhibition in class A enzymes (179).…”

Section: Inhibitory Activity Of Carbapenemsmentioning

confidence: 99%

Three Decades of β-Lactamase Inhibitors

Drawz¹,

2010

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SUMMARYSince the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamases. β-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome β-lactamase-mediated resistance, β-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner β-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of seriousEnterobacteriaceaeand penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam-β-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant β-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we review the catalytic mechanisms of each β-lactamase class. We then discuss approaches for circumventing β-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of β-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a “second generation” of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of β-lactamases.

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“…The rate constant k 3 has been shown to be the rate-determining step in imipenem hydrolysis by class A b-lactamases (NMC-A and TEM-1). 20,21 In GES b-lactamases, k 3 is rate-limiting in imipenem hydrolysis for GES-5, whereas compared with GES-1, k 2 is rate limiting. 22 Investigations are ongoing to determine the differences in hydrolysis of these carbapenems by WT KPC-2.…”

Section: Carbapenem Discriminationmentioning

confidence: 99%

Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

et al. 2010

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The molecular basis of resistance to b-lactams and b-lactam-b-lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective b-lactam therapy. Recent crystal structures of KPC-2 and other class A b-lactamases suggest that Ambler position Trp105 may be of importance in binding b-lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC-2 by conducting site-saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, -Tyr, -Asn, and -His KPC-2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC-2. Interestingly, most of the variants showed increased MICs to ampicillin-clavulanic acid but not to ampicillin-sulbactam or piperacillin-tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, -Asn, -Leu, and -Val) were studied in detail. Consistent with the MIC data, the Trp105Phe b-lactamase displayed improved catalytic efficiencies, k cat /K m , toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k cat /K m toward cefotaxime and imipenem when compared to WT b-lactamase. The Trp105Asn variant exhibited increased K m s for all substrates. In contrast, the Trp105Leu and -Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k cat /K m ) for all substrates. With respect to clavulanic acid, the K i s and partition ratios were increased for the Trp105Phe, -Asn, and -Val variants. We conclude that interactions between Trp105 of KPC-2 and the b-lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in b-lactam and b-lactamase inhibitor discrimination.

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Mechanism of turnover of imipenem by the TEM .beta.-lactamase revisited

Cited by 57 publications

References 0 publications

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of β-Lactamases

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of β-Lactamases

Three Decades of β-Lactamase Inhibitors

Elucidating the role of Trp105 in the KPC‐2 β‐lactamase

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