Isolation of a covalent intermediate in β‐lactamase I catalysis

Cartwright, Steven J.; Fink, Anthony L.

doi:10.1016/0014-5793(82)80345-1

Cited by 30 publications

(25 citation statements)

References 19 publications

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“…Similar results were obtained by Cartwright and Fink (1982) with the B. cereus I enzyme and dansyl penicillin, The expected labelled peptide \vas isolated after peptic digestion. A number of other active-site--directed inactivators of Class A enzymes are known (and at least one-penicillanic acid sulphone (Retsema et til.~1981)-is being evaluated for potential clinical use.…”

Section: Fj Lactamases· Molecular Studies 239supporting

confidence: 81%

ß-Lactamases: Molecular Studies

Coulson

1985

Biotechnology and Genetic Engineering Reviews

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Section: Fj Lactamases· Molecular Studies 239supporting

confidence: 81%

ß-Lactamases: Molecular Studies

Coulson

1985

Biotechnology and Genetic Engineering Reviews

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“…[ 14 C]benzylpenicillin (54 Ci/mol) was from Amersham International (Buckinghamshire, United Kingdom). 5Ј-Fluoresceyl-glycyl-6-aminopenicillanate (5Ј-Flu-Gly-6-APA), 5Ј-fluoresceyl-ampicillin, 6Ј-fluoresceyl-ampicillin, and dansyl-6-APA were described previously (4,23). Nonlabelled ␤-lactams were gifts from pharmaceutical companies.…”

Section: Methodsmentioning

confidence: 99%

The bimodular G57-V577 polypeptide chain of the class B penicillin-binding protein 3 of Escherichia coli catalyzes peptide bond formation from thiolesters and does not catalyze glycan chain polymerization from the lipid II intermediate

et al. 1997

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Because the specificity profile of the membrane anchor-free G57-V577 penicillin-binding protein 3 (PBP3) of Escherichia coli for a large series of ␤-lactam antibiotics is similar to that of the full-size membrane-bound PBP, the truncated PBP is expected to adopt the native folded conformation. The truncated PBP3 functions as a thiolesterase. In aqueous media and in the presence of millimolar concentrations of a properly structured amino compound, it catalyzes the aminolysis of the thiolester until completion, suggesting that the penicillinbinding module of PBP3 is designed to catalyze transpeptidation reactions. In contrast, the truncated PBP3 is devoid of glycan polymerization activity on the E. coli lipid II intermediate, suggesting that the non-penicillinbinding module of PBP3 is not a transglycosylase.The multimodular class B penicillin-binding protein 3 (PBP3) is a key element of the cell septation network in Escherichia coli (30). This tripartite protein consists of an M1-E56 membrane anchor-containing module that is fused to a G57-I237 non-penicillin-binding (n-PB) module that is fused to the D238-V577 PB module (7,12,16). The membrane anchor-free G57-V577 polypeptide chain of PBP3 which comprises the n-PB and PB modules has been overproduced in the periplasm of E. coli as an autonomous folding entity (9, 16). With "p" denoting "periplasmic," this truncated PBP3 is called PBP3p.Indirect experimental evidence suggests that the PB module of PBP3 is involved, one way or another, in peptide crosslinking during synthesis of the septal peptidoglycan (3,26). In contrast, the role of the n-PB module of PBP3 is a matter of controversy. According to Ishino and Matsuhashi (19), this module is a transglycosylase catalyzing glycan chain elongation from disaccharide (peptide) lipid II intermediates. According to van Heijenoort et al. (29), it is not.Because the thermostability and affinity for benzylpenicillin and cephalexin of the folded, membrane anchor-free polypeptide are unchanged in comparison with those of the membrane-bound PBP3, one can reasonably postulate that the conformation adopted by the truncated PBP3 reflects faithfully that of the native-state structure (9). ␤-Lactams and thiolester carbonyl donors and the disaccharide (peptide) lipid II intermediate were used to probe the enzymatic activities of the G57-V577 PBP3 and thus shed light on the possible functions of the protein in cell septation. The results are presented below.(Some of the work described in this paper is part of a dissertation presented by M.A. in partial fulfilment of a Ph.D. degree at the University of Liège.) MATERIALS AND METHODSPBP3p and carbonyl donor substrates. PBP3p was purified as described previously (9). It was stored frozen at Ϫ20°C in 10 mM Tris-HCl (pH 8.0)-10% (vol/vol) glycerol-10% (vol/vol) ethylene glycol-0.5 M NaCl. The esters and thiolesters were described previously (1, 2). [14 C]benzylpenicillin (54 Ci/mol) was from Amersham International (Buckinghamshire, United Kingdom). 5Ј-Fluoresceyl-glycyl-6-aminopenicillanate...

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“…37 h, estimated on the basis of the rate constant governing deacylation (35), reaction of the E166N mutant with substrate also results essentially in a kinetically nonturnover condition. This situation can be approximated with the catalytically competent, wt enzyme only by application of cryoenzymologic conditions (9,(47)(48)(49). In ENDOR spectroscopy the shift of each resonance feature from the Larmor frequency is a direct function of the distance between the unpaired electron of the paramagnetic nitroxyl group (50) and the corresponding class of magnetic nuclei giving rise to the resonance feature through hyperfine coupling (51).…”

Section: The Influence Of the Carbon Source On Deuterium Enrichment Omentioning

confidence: 99%

“…In this diagram, the proton ENDOR spectrum of the acylenzyme intermediate of the perdeuterated wt enzyme, formed with a kinetically specific, nitroxyl spin-labeled penicillin substrate (31,46), is compared to spectra of the acylenzyme of the E166N mutant as a function of deuterium enrichment: (a) protiated E166N in H 2 O buffer, (b) protiated E166N in perdeuterated buffer, and (c) deuterium enriched E166N in perdeuterated buffer. In these studies the reaction intermediate of the deuterium enriched wt enzyme has been kinetically isolated for spectroscopic characterization with use of organic-aqueous cosolvent mixtures in the subzero temperature range (47)(48)(49) and compared to the acylenzyme of the E166N mutant. Since the E166N mutant is deacylation impaired (21) and its acylenzyme species has a half-life at room temperature of ca.…”

Section: The Influence Of the Carbon Source On Deuterium Enrichment Omentioning

confidence: 99%