Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

American Chemical Society full text link American Chemical Society Free PMC article
Full text links

Actions

Share

.2007 Aug 7;46(31):8980-7.
doi: 10.1021/bi7006146. Epub 2007 Jul 13.

Sulbactam forms only minimal amounts of irreversible acrylate-enzyme with SHV-1 beta-lactamase

Affiliations

Sulbactam forms only minimal amounts of irreversible acrylate-enzyme with SHV-1 beta-lactamase

Monica A Totir et al. Biochemistry..

Abstract

Sulbactam is a mechanism-based inhibitor of beta-lactamase enzymes used in clinical practice. It undergoes a complex series of chemical reactions in the active site that have been studied extensively in the past three decades. However, the actual species that gives rise to inhibition in a clinical setting has not been established. Recent studies by our group, using Raman microscopy and X-ray crystallography, have found that large quantities of enamine-based acyl-enzyme species are present within minutes in single crystals of SHV-1 beta-lactamases which can lead to significant inhibition. The enamines are formed by breakdown of the cyclic beta-lactam structures with further transformations leading to imine formation and subsequent isomerization to cis and/or trans enamines. Another favored form of inhibition arises from attack on the imine by a second nucleophilic amino acid side chain, e.g., from serine 130, to form a cross-linked species in the active site that can degrade to an acrylate-like species irreversibly bound to the enzyme. Thus, the imine is at a branch point on the reaction pathway. Using sulbactam and 6,6-dideuterated sulbactam we follow these alternate paths in WT and E166A SHV-1 beta-lactamase by means of Raman microscopic studies on single enzyme crystals. For the unlabeled sulbactam, the Raman data show the presence of an acrylate-like species, probably 3-serine acrylate, several hours after the reaction is started in the crystal. However, for the 6,6-dideutero analogue the acrylate signature appears on the time scale of minutes. The Raman signatures, principally an intense feature near 1530 cm-1, are assigned based on quantum mechanical calculations on model compounds that mimic acrylate species in the active site. The different time scales observed for acrylate-like product formation are ascribed to different rates of reaction involving the imine intermediate. It is proposed that for the unsubstituted sulbactam the conversion from imine to enamine, which involves breaking a C-H bond, is aided by quantum mechanical tunneling. For the 6,6-dideutero-sulbactam the same step involves breaking a C-D bond, which has little or no assistance from tunneling. Consequently the conversion to enamines is slower, and a higher population of imine results, presenting the opportunity for the competing reaction with the second nucleophile, serine 130 being the prime candidate. The hydrolysis of the resulting cross-linked intermediate leads to the observed rapid buildup of the acrylate product in the Raman spectra from the dideutero analogue. The protocol used here, essentially running the reactions with the two forms of sulbactam in parallel, provides an element of control and enables us to conclude that, for the unsubstituted sulbactam, the formation of the cross-linked intermediate and the final irreversible acrylate product is not a significant route to inhibition of SHV-1.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Class A β-lactamase inhibitors
Figure 2
Figure 2
Raman difference spectra of E166A SHV-1 β-lactamase reacting with sulbactam 5mM at different time points. The peak near 1045 cm−1 is due to HEPES buffer.
Figure 3
Figure 3
Time dependence of the 1530 cm−1 feature height given by sulbactam reacting inside an E166A SHV-1 β-lactamase crystal. The estimated standard deviation in the intensity values is ± 12%.
Figure 4
Figure 4
Calculated Raman spectrum of 3-methoxyacrylate, obtained by using the Gaussian 03 software, where the main features are; 1629 cm−1 C=O and C=C stretch, 1536 cm−1 C=C stretch and delocalized motions over the entire chain, 1330 cm−1 C=C stretch and C-H bending, 1240 cm−1 C=C and C=O stretch, C-H bending
Figure 5
Figure 5
Raman difference spectra of E166A SHV-1 β-lactamase reacting with 6,6 dideuterated sulbactam 10 mM at different time points
Figure 6
Figure 6
Time dependence of the 1530 cm−1 feature height given by 6, 6 dideuterated sulbactam reacting inside an E166A SHV-1 β-lactamase crystal. The estimated standard deviation in the intensity values is ± 12%.
Figure 7
Figure 7
Raman difference spectra of wild type SHV-1 β-lactamase reacting with sulbactam (red) at 75 minutes or 6,6 dideuterated sulbactam (green) at 80 minutes
Figure 8
Figure 8
Time dependence of the 1502 cm−1 feature height given by 6, 6 dideuterated sulbactam reacting inside a WT SHV-1 β-lactamase crystal. The estimated standard deviation in the intensity values is ± 12%.
Scheme 1
Scheme 1
Simplified reaction mechanism for Class A β-lactamase with sulbactam
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Helfand M, Bonomo R. β-Lactamases: A Survey of Protein Diversity. Current Drug Targets. 2003;3:9–23. - PubMed
    1. Brenner DG, Knowles JR. Penicillanic Acid Sulfone: An Unexpected Isotope Effect in the Interaction of 6a- and 6b-Monodeuterio and of 6,6-Dideuterio Derivatives with RTEM β-Lactamase from Escherichia coli. Biochemistry. 1981;20:3680–3687. - PubMed
    1. Bonomo RA, Dawes CG, Knox JR, Shlaes DM. β-Lactamase mutations far from the active site influence inhibitor binding. Biochimica et Biophysica Acta. 1995;1247:121–125. - PubMed
    1. Bush K, Clarissa M, Rasmussen BA, Lee VJ, Yang Y. Kinetic Interactions of Tazobactam with β-Lactamases from All Major Structural Classes. Antimicrobial Agents and Chemotherapy. 1993;37:851–858. - PMC - PubMed
    1. Brown RPA, Aplin RT, Schofield CJ. Inhibition of TEM-2 β-Lactamase from Escherichia coli by Clavulanic Acid: of Intermediates by Electrospray Ionization Mass Spectrometry. Biochemistry. 1996;35:12421–12432. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
American Chemical Society full text link American Chemical Society Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp