X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine
BACKGROUND: Para-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs. METHODS: Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-acti...
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ftpubmed:oai:pubmedcentral.nih.gov:7569297 2023-05-15T13:46:52+02:00 X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine Ásgeirsson, Bjarni Markússon, Sigurbjörn Hlynsdóttir, Sigríður S. Helland, Ronny Hjörleifsson, Jens G. 2020-10-15 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569297/ https://doi.org/10.1016/j.bbrep.2020.100830 en eng Elsevier http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569297/ http://dx.doi.org/10.1016/j.bbrep.2020.100830 © 2020 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). CC-BY-NC-ND Biochem Biophys Rep Research Article Text 2020 ftpubmed https://doi.org/10.1016/j.bbrep.2020.100830 2020-10-25T00:48:30Z BACKGROUND: Para-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs. METHODS: Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-active Vibrio AP (VAP) was performed and the structure solved. RESULTS: Inhibition of VAP fitted a non-competitive kinetic model (K(m) unchanged, V(max) reduced) with IC(50) 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC(50) 27.9 mM at pH 8.0, where the addition of 0.5 M NaCl altered the inhibition to the level observed at pH 9.8. APs from E. coli and calf intestines were less sensitive to cyclohexylamine, whereas an Antarctic bacterial AP was similar to VAP in this respect. X-ray crystallography at 2.3 Å showed two binding sites, one in the active site channel and another at the surface close to dimer interface. Antarctic bacterial AP and VAP have Trp274 in common in their active-sites, that takes part in binding cyclohexylamine. VAP variants W274A, W274K, and W274H gave IC(50) values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8. CONCLUSIONS: The binding of cyclohexylamine in locations at the dimeric interface and/or in the active site of APs may delay product release or reduce the rate of catalytic step(s) involving conformational changes and intersubunit communications. GENERAL SIGNIFICANCE: Cyclohexylamine is a common chemical in industries and used as a counterion in substrates for alkaline phosphatase, a clinically important and common enzyme in the biosphere. Text Antarc* Antarctic PubMed Central (PMC) Antarctic Biochemistry and Biophysics Reports 24 100830 |
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Research Article Ásgeirsson, Bjarni Markússon, Sigurbjörn Hlynsdóttir, Sigríður S. Helland, Ronny Hjörleifsson, Jens G. X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
topic_facet |
Research Article |
description |
BACKGROUND: Para-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs. METHODS: Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-active Vibrio AP (VAP) was performed and the structure solved. RESULTS: Inhibition of VAP fitted a non-competitive kinetic model (K(m) unchanged, V(max) reduced) with IC(50) 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC(50) 27.9 mM at pH 8.0, where the addition of 0.5 M NaCl altered the inhibition to the level observed at pH 9.8. APs from E. coli and calf intestines were less sensitive to cyclohexylamine, whereas an Antarctic bacterial AP was similar to VAP in this respect. X-ray crystallography at 2.3 Å showed two binding sites, one in the active site channel and another at the surface close to dimer interface. Antarctic bacterial AP and VAP have Trp274 in common in their active-sites, that takes part in binding cyclohexylamine. VAP variants W274A, W274K, and W274H gave IC(50) values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8. CONCLUSIONS: The binding of cyclohexylamine in locations at the dimeric interface and/or in the active site of APs may delay product release or reduce the rate of catalytic step(s) involving conformational changes and intersubunit communications. GENERAL SIGNIFICANCE: Cyclohexylamine is a common chemical in industries and used as a counterion in substrates for alkaline phosphatase, a clinically important and common enzyme in the biosphere. |
format |
Text |
author |
Ásgeirsson, Bjarni Markússon, Sigurbjörn Hlynsdóttir, Sigríður S. Helland, Ronny Hjörleifsson, Jens G. |
author_facet |
Ásgeirsson, Bjarni Markússon, Sigurbjörn Hlynsdóttir, Sigríður S. Helland, Ronny Hjörleifsson, Jens G. |
author_sort |
Ásgeirsson, Bjarni |
title |
X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
title_short |
X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
title_full |
X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
title_fullStr |
X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
title_full_unstemmed |
X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
title_sort |
x-ray crystal structure of vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
publisher |
Elsevier |
publishDate |
2020 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569297/ https://doi.org/10.1016/j.bbrep.2020.100830 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Biochem Biophys Rep |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569297/ http://dx.doi.org/10.1016/j.bbrep.2020.100830 |
op_rights |
© 2020 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
op_rightsnorm |
CC-BY-NC-ND |
op_doi |
https://doi.org/10.1016/j.bbrep.2020.100830 |
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Biochemistry and Biophysics Reports |
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24 |
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100830 |
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1766245498540261376 |