Different transition-state structures for the reactions of β-lactams and analogous β-sultams with serine β-lactamases

W.Y. Tsang, N. Ahmed, P.S. Hinchliffe, J.M. Wood, L.P. Harding, A.P. Laws, M.I. Page

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

β-Sultams are the sulfonyl analogues of β-lactams, and N-acyl β-sultams are novel inactivators of the class C β-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C−O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl β-lactams are substrates for β-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation−deacylation process. The rates of acylation of the enzyme by the β-lactams, measured by the second-order rate constant for hydrolysis, kcat/Km, and those of sulfonylation by the β-sultams, measured by the second-order rate constant for inactivation, ki, both show a similar pH dependence to that exhibited by the β-lactamase-catalyzed hydrolysis of β-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl β-lactams increase the rate of alkaline hydrolysis and give a Bronsted βlg of −0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted βlg for the β-lactamase-catalyzed hydrolysis of the same substrates is −0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted βlg for the alkaline hydrolysis of N-aroyl β-sultams is −0.73, similar to that for the β-lactams, that for the sulfonylation of β-lactamase by these compounds is −1.46, compatible with significant amide anion expulsion/S−N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the β-sultam.
Original languageEnglish
Pages (from-to)17556-17564
Number of pages9
JournalJournal of the American Chemical Society
Volume127
Issue number49
DOIs
Publication statusPublished - 18 Nov 2005

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Lactams
Serine
Hydrolysis
Enzymes
Anions
Rate constants
Negative ions
Enterobacter cloacae
Acylation
Electrospray ionization
Antibiotics
Substrates
Carbon Monoxide
naphthosultone
Amides
Mass Spectrometry
Catalytic Domain
Esters
Spectroscopy
Electrons

Cite this

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title = "Different transition-state structures for the reactions of β-lactams and analogous β-sultams with serine β-lactamases",
abstract = "β-Sultams are the sulfonyl analogues of β-lactams, and N-acyl β-sultams are novel inactivators of the class C β-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C−O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl β-lactams are substrates for β-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation−deacylation process. The rates of acylation of the enzyme by the β-lactams, measured by the second-order rate constant for hydrolysis, kcat/Km, and those of sulfonylation by the β-sultams, measured by the second-order rate constant for inactivation, ki, both show a similar pH dependence to that exhibited by the β-lactamase-catalyzed hydrolysis of β-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl β-lactams increase the rate of alkaline hydrolysis and give a Bronsted βlg of −0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted βlg for the β-lactamase-catalyzed hydrolysis of the same substrates is −0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted βlg for the alkaline hydrolysis of N-aroyl β-sultams is −0.73, similar to that for the β-lactams, that for the sulfonylation of β-lactamase by these compounds is −1.46, compatible with significant amide anion expulsion/S−N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the β-sultam.",
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Different transition-state structures for the reactions of β-lactams and analogous β-sultams with serine β-lactamases. / Tsang, W.Y.; Ahmed, N.; Hinchliffe, P.S.; Wood, J.M.; Harding, L.P.; Laws, A.P.; Page, M.I.

In: Journal of the American Chemical Society, Vol. 127, No. 49, 18.11.2005, p. 17556-17564.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Different transition-state structures for the reactions of β-lactams and analogous β-sultams with serine β-lactamases

AU - Tsang, W.Y.

AU - Ahmed, N.

AU - Hinchliffe, P.S.

AU - Wood, J.M.

AU - Harding, L.P.

AU - Laws, A.P.

AU - Page, M.I.

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N2 - β-Sultams are the sulfonyl analogues of β-lactams, and N-acyl β-sultams are novel inactivators of the class C β-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C−O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl β-lactams are substrates for β-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation−deacylation process. The rates of acylation of the enzyme by the β-lactams, measured by the second-order rate constant for hydrolysis, kcat/Km, and those of sulfonylation by the β-sultams, measured by the second-order rate constant for inactivation, ki, both show a similar pH dependence to that exhibited by the β-lactamase-catalyzed hydrolysis of β-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl β-lactams increase the rate of alkaline hydrolysis and give a Bronsted βlg of −0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted βlg for the β-lactamase-catalyzed hydrolysis of the same substrates is −0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted βlg for the alkaline hydrolysis of N-aroyl β-sultams is −0.73, similar to that for the β-lactams, that for the sulfonylation of β-lactamase by these compounds is −1.46, compatible with significant amide anion expulsion/S−N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the β-sultam.

AB - β-Sultams are the sulfonyl analogues of β-lactams, and N-acyl β-sultams are novel inactivators of the class C β-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C−O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl β-lactams are substrates for β-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation−deacylation process. The rates of acylation of the enzyme by the β-lactams, measured by the second-order rate constant for hydrolysis, kcat/Km, and those of sulfonylation by the β-sultams, measured by the second-order rate constant for inactivation, ki, both show a similar pH dependence to that exhibited by the β-lactamase-catalyzed hydrolysis of β-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl β-lactams increase the rate of alkaline hydrolysis and give a Bronsted βlg of −0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted βlg for the β-lactamase-catalyzed hydrolysis of the same substrates is −0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted βlg for the alkaline hydrolysis of N-aroyl β-sultams is −0.73, similar to that for the β-lactams, that for the sulfonylation of β-lactamase by these compounds is −1.46, compatible with significant amide anion expulsion/S−N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the β-sultam.

U2 - 10.1021/ja056124z

DO - 10.1021/ja056124z

M3 - Article

VL - 127

SP - 17556

EP - 17564

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

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