The roles of the carboxy group in β-lactam antibiotics and lysine 234 in β-lactamase I

Laws, Andrew P.; Layland, Nicola J.; Proctor, David G.; Page, Michael I.

doi:10.1039/p29930000017

Cited by 14 publications

(16 citation statements)

References 29 publications

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“…The results obtained with the modified substrates can be most significantly analysed by making use of the 'Enzyme Rate Enhancement Factor' (EREF) introduced by Laws and Page (1989), which represents the ratio of the kcat/Km value of the enzymic reaction to the second-order rate constant, koH, observed for the alkaline hydrolysis of the fi-lactam, which reflects the intrinsic susceptibility of the compound to a nucleophilic attack. In agreement with previous results (Laws and Page, 1989;Varetto et al, 1991;Laws et al, 1993), and despite the fact that the negative electrostatic potential maps for the benzylpenicillin Laws and Page (1989). § Results for benzyldeacetyl cephalosporanic acid and its lactone (Laws et al, 1993).…”

Section: Discussionsupporting

confidence: 89%

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Catalytic mechanism of active-site serine β-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad

Dubus

Wilkin

Raquet

et al. 1994

Biochemical Journal

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The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.

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Section: Discussionsupporting

confidence: 89%

“…In agreement with previous results (Laws and Page, 1989;Varetto et al, 1991;Laws et al, 1993), and despite the fact that the negative electrostatic potential maps for the benzylpenicillin Laws and Page (1989). § Results for benzyldeacetyl cephalosporanic acid and its lactone (Laws et al, 1993).…”

Section: Discussionsupporting

confidence: 89%

Catalytic mechanism of active-site serine β-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad

Dubus

Wilkin

Raquet

et al. 1994

Biochemical Journal

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“…The ester group is more electron-withdrawing than the carboxylate group, which increases both the electrophilicity of the b-lactam carbonyl carbon and the leaving-group ability of the b-lactam amino group. 7,12 This electronwithdrawing e ect is re¯ected in the shift of the amine pK a from 5.2 for benzylpenicilloic acid to 3.2 for its methyl ester. 11 Useful esters of b-lactam antibiotics have thus to feature the following kinetic characteristics:…”

Section: Resultsmentioning

confidence: 99%

“…The white needles formed were collected, washed with water and dried over P 2 O 5 (yield: 1.17 g, 100%). Mp 157±159 C. 1 Cyclohexylidene-N-bromomethylsuccinimide (12). A suspension of the former compound (0.4 g, 2 mmol), phosphorus tribromide (0.52 g, 2 mmol) and toluene (2 mL) was re¯uxed for 4 h. The solution was left to settle and the supernatant was poured out.…”

Section: Cyclohexylidene-n-hydroxymethylsuccinimide (11)mentioning

confidence: 99%

Syntheses and hydrolysis of basic and dibasic ampicillin esters tailored for intracellular accumulation

Paternotte

Fan

Scrève

et al. 2001

Bioorganic & Medicinal Chemistry

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AbstractÐReadily hydrolysable basic and dibasic esters of ampicillin were synthesised by alkylation of the carboxylate function of ampicillin to obtain prodrugs that may accumulate in cells and allow for an intracellular delivery of ampicillin (Fan et al., Bioorg. Med. Chem. Lett. 1997, 7, 3107). We found that the b-lactam ring cleavage and the hydrolysis of the ester function were competitive reactions. The prerequisite for biological activity of compounds of this type is therefore that ester hydrolysis proceeds faster than ring opening. Some synthesised compounds show promise as prodrugs since they displayed a reasonable stability and regenerate large quantities of bioactive ampicillin in broth. #

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“…Hydrolysis of the substrate was followed by measuring the decrease in absorbance at 260 and 230 nm for cephaloridine and benzylpenicillin respectively as a function of time. The kinetic parameters were determined as described previously [33] and curve fitting was achieved using ENZFITTER (Elsevier Biosoft, Cambridge, U.K.) or SCIENTIST (MicroMath Scientific Software, Salt Lake City, UT, U.S.A.) software.…”

Section: Kinetic Studiesmentioning

confidence: 99%

The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase

Bounaga

Laws

Galleni

et al. 1998

Biochemical Journal

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171

229

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The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM. The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5. The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

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The roles of the carboxy group in β-lactam antibiotics and lysine 234 in β-lactamase I

Cited by 14 publications

References 29 publications

Catalytic mechanism of active-site serine β-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad

Catalytic mechanism of active-site serine β-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad

Syntheses and hydrolysis of basic and dibasic ampicillin esters tailored for intracellular accumulation

The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase

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