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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ftdoajarticles:oai:doaj.org/article:76850bdaa0ea4fb387b62e19553df988 2023-05-15T13:48:56+02:00 X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine Bjarni Ásgeirsson Sigurbjörn Markússon Sigríður S. Hlynsdóttir Ronny Helland Jens G. Hjörleifsson 2020-12-01T00:00:00Z https://doi.org/10.1016/j.bbrep.2020.100830 https://doaj.org/article/76850bdaa0ea4fb387b62e19553df988 EN eng Elsevier http://www.sciencedirect.com/science/article/pii/S2405580820301400 https://doaj.org/toc/2405-5808 2405-5808 doi:10.1016/j.bbrep.2020.100830 https://doaj.org/article/76850bdaa0ea4fb387b62e19553df988 Biochemistry and Biophysics Reports, Vol 24, Iss , Pp 100830- (2020) Enzymology Alkaline phosphatase Enzyme inhibitor P-nitrophenyl phosphate Cyclohexylamine Non-competitive Biology (General) QH301-705.5 Biochemistry QD415-436 article 2020 ftdoajarticles https://doi.org/10.1016/j.bbrep.2020.100830 2022-12-31T04:51:20Z 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 (Km unchanged, Vmax reduced) with IC50 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC50 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 IC50 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 Directory of Open Access Journals: DOAJ Articles Antarctic Biochemistry and Biophysics Reports 24 100830 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Enzymology Alkaline phosphatase Enzyme inhibitor P-nitrophenyl phosphate Cyclohexylamine Non-competitive Biology (General) QH301-705.5 Biochemistry QD415-436 |
spellingShingle |
Enzymology Alkaline phosphatase Enzyme inhibitor P-nitrophenyl phosphate Cyclohexylamine Non-competitive Biology (General) QH301-705.5 Biochemistry QD415-436 Bjarni Ásgeirsson Sigurbjörn Markússon Sigríður S. Hlynsdóttir Ronny Helland Jens G. Hjörleifsson X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine |
topic_facet |
Enzymology Alkaline phosphatase Enzyme inhibitor P-nitrophenyl phosphate Cyclohexylamine Non-competitive Biology (General) QH301-705.5 Biochemistry QD415-436 |
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 (Km unchanged, Vmax reduced) with IC50 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC50 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 IC50 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 |
Bjarni Ásgeirsson Sigurbjörn Markússon Sigríður S. Hlynsdóttir Ronny Helland Jens G. Hjörleifsson |
author_facet |
Bjarni Ásgeirsson Sigurbjörn Markússon Sigríður S. Hlynsdóttir Ronny Helland Jens G. Hjörleifsson |
author_sort |
Bjarni Ásgeirsson |
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://doi.org/10.1016/j.bbrep.2020.100830 https://doaj.org/article/76850bdaa0ea4fb387b62e19553df988 |
geographic |
Antarctic |
geographic_facet |
Antarctic |
genre |
Antarc* Antarctic |
genre_facet |
Antarc* Antarctic |
op_source |
Biochemistry and Biophysics Reports, Vol 24, Iss , Pp 100830- (2020) |
op_relation |
http://www.sciencedirect.com/science/article/pii/S2405580820301400 https://doaj.org/toc/2405-5808 2405-5808 doi:10.1016/j.bbrep.2020.100830 https://doaj.org/article/76850bdaa0ea4fb387b62e19553df988 |
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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1766249979787083776 |