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dc.contributor.authorÁsgeirsson, Bjarni
dc.contributor.authorMarkússon, Sigurbjörn
dc.contributor.authorHlynsdóttir, Sigríður S.
dc.contributor.authorHelland, Ronny
dc.contributor.authorHjörleifsson, Jens G.
dc.date.accessioned2021-01-21T07:44:05Z
dc.date.available2021-01-21T07:44:05Z
dc.date.issued2020-10-15
dc.description.abstract<i>Background</i> - <i>Para</i>-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.<br><br> <i>Methods</i> - Cyclohexylamine inhibited four different APs. Co-crystallization with the cold-active <i>Vibrio</i> AP (VAP) was performed and the structure solved.<br><br> <i>Results</i> - Inhibition of VAP fitted a non-competitive kinetic model (K<sub>m</sub> unchanged, V<sub>max</sub> reduced) with IC<sub>50</sub> 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC<sub>50</sub> 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 <i>E. coli</i> 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<sub>50</sub> values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8.<br><br> <i>Conclusions</i> - 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.<br><br> <i>General significance</i> - 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.en_US
dc.identifier.citationÁsgeirsson, Markússon, Hlynsdóttir, Helland R, Hjörleifsson. X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine. Biochemistry and Biophysics Reports. 2020;24:100830en_US
dc.identifier.cristinIDFRIDAID 1842773
dc.identifier.doihttps://doi.org/10.1016/j.bbrep.2020.100830
dc.identifier.issn2405-5808
dc.identifier.urihttps://hdl.handle.net/10037/20343
dc.language.isoengen_US
dc.publisherElsevieren_US
dc.relation.journalBiochemistry and Biophysics Reports
dc.rights.accessRightsopenAccessen_US
dc.rights.holderCopyright 2020 The Author(s)en_US
dc.subjectVDP::Mathematics and natural science: 400::Chemistry: 440en_US
dc.subjectVDP::Matematikk og Naturvitenskap: 400::Kjemi: 440en_US
dc.titleX-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamineen_US
dc.type.versionpublishedVersionen_US
dc.typeJournal articleen_US
dc.typeTidsskriftartikkelen_US
dc.typePeer revieweden_US


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