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-a...
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ftunivtroemsoe:oai:munin.uit.no:10037/20343 2023-05-15T13:52:48+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 https://hdl.handle.net/10037/20343 https://doi.org/10.1016/j.bbrep.2020.100830 eng eng Elsevier Biochemistry and Biophysics Reports Ásgeirsson, Markússon, Hlynsdóttir, Helland R, Hjörleifsson. X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine. Biochemistry and Biophysics Reports. 2020;24:100830 FRIDAID 1842773 https://doi.org/10.1016/j.bbrep.2020.100830 2405-5808 https://hdl.handle.net/10037/20343 openAccess Copyright 2020 The Author(s) VDP::Mathematics and natural science: 400::Chemistry: 440 VDP::Matematikk og Naturvitenskap: 400::Kjemi: 440 Journal article Tidsskriftartikkel Peer reviewed publishedVersion 2020 ftunivtroemsoe https://doi.org/10.1016/j.bbrep.2020.100830 2021-06-25T17:57:53Z 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. Article in Journal/Newspaper Antarc* Antarctic University of Tromsø: Munin Open Research Archive Antarctic Biochemistry and Biophysics Reports 24 100830 |
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Open Polar |
collection |
University of Tromsø: Munin Open Research Archive |
op_collection_id |
ftunivtroemsoe |
language |
English |
topic |
VDP::Mathematics and natural science: 400::Chemistry: 440 VDP::Matematikk og Naturvitenskap: 400::Kjemi: 440 |
spellingShingle |
VDP::Mathematics and natural science: 400::Chemistry: 440 VDP::Matematikk og Naturvitenskap: 400::Kjemi: 440 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 |
VDP::Mathematics and natural science: 400::Chemistry: 440 VDP::Matematikk og Naturvitenskap: 400::Kjemi: 440 |
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 |
Article in Journal/Newspaper |
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 |
https://hdl.handle.net/10037/20343 https://doi.org/10.1016/j.bbrep.2020.100830 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_relation |
Biochemistry and Biophysics Reports Ásgeirsson, Markússon, Hlynsdóttir, Helland R, Hjörleifsson. X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine. Biochemistry and Biophysics Reports. 2020;24:100830 FRIDAID 1842773 https://doi.org/10.1016/j.bbrep.2020.100830 2405-5808 https://hdl.handle.net/10037/20343 |
op_rights |
openAccess Copyright 2020 The Author(s) |
op_doi |
https://doi.org/10.1016/j.bbrep.2020.100830 |
container_title |
Biochemistry and Biophysics Reports |
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24 |
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100830 |
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1766257560885657600 |