Inactivation of class A .beta.-lactamases by clavulanic acid: the role of arginine-244 in a proposed nonconcerted sequence of events

Imtiaz, Uzma; Billings, Eric M.; Knox, James R.; Manavathu, Elias K.; Lerner, Stephen A.; Mobashery, Shahriar

doi:10.1021/ja00064a003

Cited by 137 publications

(195 citation statements)

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“…According to the Hammond postulate, this implies a later transition state along the reaction coordinate with significant negative charge build-up, thereby being more susceptible to the electronic effects of the C-4 substituents. The ρ I value determined here for the monobactams 5 is higher than the corresponding ρ I value of 1.35 reported for the alkaline hydrolysis of cephalosporins, 42 a difference that almost certainly reflects the 9 shorter distance between the electron-withdrawing substituents and the nitrogen atom in the β-lactam scaffold of 5.…”

Section: Alkaline Hydrolysiscontrasting

confidence: 76%

“…8 Among the most extensively studied enzymatic reactions of β-lactams is the inhibition of class A β-lactamases by penam sulfone inhibitors such as sulbactam, 1 (Scheme 1), tazobactam and their analogues. [9][10][11][12][13][14][15] Reaction of penam sulfones with the catalytic Ser70 involves β-lactam ringopening and leads to the departure of a sulfinate leaving group from C-5 and formation of an imine (Scheme 1). 1,14 The imine then undergoes a cascade of reactions including nucleophilic attack by Ser130 hydroxyl group to form a stable acrylate ester capable of preventing hydrolysis of the acylenzyme, and thus leading to irreversible inhibition.…”

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confidence: 99%

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The efficiency of C-4 substituents in activating the β-lactam scaffold towards serine proteases and hydroxide ion

et al. 2007

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Section: Alkaline Hydrolysiscontrasting

confidence: 76%

mentioning

confidence: 99%

The efficiency of C-4 substituents in activating the β-lactam scaffold towards serine proteases and hydroxide ion

et al. 2007

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“…The major effect of the S130G mutation on apparent K m and K I emphasizes the precise topological role of this amino acid (17,(22)(23)(24)(25)(26)(27). A direct consequence of the Ser 3 Gly substitution is reflected by the amount of S130G versus SHV-1 inhibited in 20 min, the time required for E. coli cell division.…”

Section: Discussionmentioning

confidence: 99%

“…The kinetic experiments and analyses were patterned after the methods of Imtiaz et al (17) and will be described briefly below.…”

Section: Determination Of Steady-state Expression Using An Enzyme-linkedmentioning

confidence: 99%

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Helfand

Bethel

Hujer

et al. 2003

Journal of Biological Chemistry

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Bacterial resistance to ␤-lactam/␤-lactamase inhibitor combinations by single amino acid mutations in class A ␤-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A ␤-lactamases, alterations at Ser-130 confer resistance to inactivation by the ␤-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 ␤-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G ␤-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G ␤-lactamase demonstrated a significant elevation in apparent K m and a reduction in k cat /K m for ampicillin. Marked increases in the dissociation constant for the preacylation complex, K I , of clavulanic acid (SHV-1, 0.14 M; S130G, 46.5 M) and tazobactam (SHV-1, 0.07 M; S130G, 4.2 M) were observed. In contrast, the k inact s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for ␤-lactams and ␤-lactamase inhibitors. These results underscore the mechanistic versatility of class A ␤-lactamases and have implications for the design of novel ␤-lactamase inhibitors.

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“…that Arg-244 coordinates a water molecule with the backbone carbonyl of Val-216 (10,15,16). This water molecule is postulated to play a key role in inhibitor affinity and provide the proton source essential for terminal inactivation of the enzyme ( Fig.…”

mentioning

confidence: 99%

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

Thomson

Distler

Prati

et al. 2006

Journal of Biological Chemistry

109

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Inhibitor-resistant class A ␤-lactamases are an emerging threat to the use of ␤-lactam/␤-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A ␤-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A ␤-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A ␤-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/ clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E ␤-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate k inact for all mutants. Electrospray ionization mass spectrometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C 3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV ␤-lactamase.␤-Lactams have been the cornerstone of antibacterial chemotherapy since the introduction of penicillin. Although this family of antibiotics now contains numerous classes (penicillins, cephalosporins, and carbapenems, see Fig. 1), their use is threatened by the expansion and spread of ␤-lactamase enzymes (1). These bacterial enzymes hydrolyze ␤-lactam antibiotics before reaching their target, the penicillin-binding proteins. In an effort to retain the utility of several generations of life-saving ␤-lactam antibiotics, ␤-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) have been developed. These mechanism-based inhibitors are ␤-lactam compounds that experience multistep reactions within the active site of ␤-lactamase enzymes (2-4). Typically the inhibitors lack antimicrobial activity and are formulated with a ␤-lactam antibiotic to act as shields against ␤-lactamase enzymes. This allows the ␤-lactam to bypass the ␤-lactamase and inactivate the penicillin binding proteins.Unfortunately, inhibitor-resistant class A ␤-lactamases are emerging in the clinic and undermining the use of ␤-lactam/␤-lactamase inhibitor therapy (5-7). Interest has been renewed in the discovery of novel ␤-lactamase inactivators to circumvent these inhibitor-resistant enzymes (8 -11). In this context, detailed analy...

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Inactivation of class A .beta.-lactamases by clavulanic acid: the role of arginine-244 in a proposed nonconcerted sequence of events

Cited by 137 publications

References 1 publication

The efficiency of C-4 substituents in activating the β-lactam scaffold towards serine proteases and hydroxide ion

The efficiency of C-4 substituents in activating the β-lactam scaffold towards serine proteases and hydroxide ion

Understanding Resistance to β-Lactams and β-Lactamase Inhibitors in the SHV β-Lactamase

Probing Active Site Chemistry in SHV β-Lactamase Variants at Ambler Position 244

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