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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Article number100830
JournalBiochemistry and Biophysics Reports
Volume24
DOIs
Publication statusPublished - Dec 2020

Bibliographical note

Funding Information:
Financial support from the Icelandic Centre for Research Fund (project 141619?053) and the Science Institute of the University of Iceland is gratefully acknowledged. The authors also thank Dr. Bjarte Aarmo Lund, Department of Chemistry, Faculty of Science and Technology at UiT ? The Arctic University of Norway, NO-9037 Troms?, Norway, for expert assistance with protein crystallographic data collection, refinement and analyses. Furthermore, the authors would like to thank Prof. Petri Kursula and Dr. Arne Raasakka, Department of Biomedicine, University of Bergen, for their help with crystallographic refinement and data processing. The authors further acknowledge the use of beamtime at the BioMAX beamline at MAX IV Laboratory in Lund, SE with gratitude.

Funding Information:
Financial support from the Icelandic Centre for Research Fund (project 141619–053) and the Science Institute of the University of Iceland is gratefully acknowledged. The authors also thank Dr. Bjarte Aarmo Lund, Department of Chemistry, Faculty of Science and Technology at UiT – The Arctic University of Norway, NO-9037 Tromsø, Norway, for expert assistance with protein crystallographic data collection, refinement and analyses. Furthermore, the authors would like to thank Prof. Petri Kursula and Dr. Arne Raasakka, Department of Biomedicine, University of Bergen, for their help with crystallographic refinement and data processing. The authors further acknowledge the use of beamtime at the BioMAX beamline at MAX IV Laboratory in Lund, SE with gratitude.

Publisher Copyright:
© 2020 The Author(s)

Other keywords

  • Alkaline phosphatase
  • Cyclohexylamine
  • Enzyme inhibitor
  • Enzymology
  • Non-competitive
  • P-nitrophenyl phosphate

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