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Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone

[Image: see text] For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH(2)OH group (6β-(hydroxymethyl)penicillanic acid). The 6β substitution improves both in...

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Autores principales: Che, Tao, Rodkey, Elizabeth A., Bethel, Christopher R., Shanmugam, Sivaprakash, Ding, Zhe, Pusztai-Carey, Marianne, Nottingham, Michael, Chai, Weirui, Buynak, John D., Bonomo, Robert A., van den Akker, Focco, Carey, Paul R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310624/
https://www.ncbi.nlm.nih.gov/pubmed/25536850
http://dx.doi.org/10.1021/bi501197t
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author Che, Tao
Rodkey, Elizabeth A.
Bethel, Christopher R.
Shanmugam, Sivaprakash
Ding, Zhe
Pusztai-Carey, Marianne
Nottingham, Michael
Chai, Weirui
Buynak, John D.
Bonomo, Robert A.
van den Akker, Focco
Carey, Paul R.
author_facet Che, Tao
Rodkey, Elizabeth A.
Bethel, Christopher R.
Shanmugam, Sivaprakash
Ding, Zhe
Pusztai-Carey, Marianne
Nottingham, Michael
Chai, Weirui
Buynak, John D.
Bonomo, Robert A.
van den Akker, Focco
Carey, Paul R.
author_sort Che, Tao
collection PubMed
description [Image: see text] For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH(2)OH group (6β-(hydroxymethyl)penicillanic acid). The 6β substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a β-aminoacrylate) in 1 h for sulbactam and in 4 h for 6β-(hydroxymethyl) sulbactam. Rapid mix–rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6β-(hydroxymethyl) sulbactam formed longer-lived acyl–enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6β-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6β-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis.
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spelling pubmed-43106242015-12-23 Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone Che, Tao Rodkey, Elizabeth A. Bethel, Christopher R. Shanmugam, Sivaprakash Ding, Zhe Pusztai-Carey, Marianne Nottingham, Michael Chai, Weirui Buynak, John D. Bonomo, Robert A. van den Akker, Focco Carey, Paul R. Biochemistry [Image: see text] For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH(2)OH group (6β-(hydroxymethyl)penicillanic acid). The 6β substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a β-aminoacrylate) in 1 h for sulbactam and in 4 h for 6β-(hydroxymethyl) sulbactam. Rapid mix–rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6β-(hydroxymethyl) sulbactam formed longer-lived acyl–enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6β-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6β-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis. American Chemical Society 2014-12-23 2015-01-27 /pmc/articles/PMC4310624/ /pubmed/25536850 http://dx.doi.org/10.1021/bi501197t Text en Copyright © 2014 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Che, Tao
Rodkey, Elizabeth A.
Bethel, Christopher R.
Shanmugam, Sivaprakash
Ding, Zhe
Pusztai-Carey, Marianne
Nottingham, Michael
Chai, Weirui
Buynak, John D.
Bonomo, Robert A.
van den Akker, Focco
Carey, Paul R.
Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone
title Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone
title_full Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone
title_fullStr Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone
title_full_unstemmed Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone
title_short Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone
title_sort detecting a quasi-stable imine species on the reaction pathway of shv-1 β-lactamase and 6β-(hydroxymethyl)penicillanic acid sulfone
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310624/
https://www.ncbi.nlm.nih.gov/pubmed/25536850
http://dx.doi.org/10.1021/bi501197t
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