6t26

X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine

spinqualitylabelsall models 6t26, resolution 2.27Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6t26 is a 2 chain structure with sequence from Vibrio sp. G15-21. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.265Å Ligands: CL, HAI, MG, PO4, ZN Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function Q93P54_9VIBR Publication Abstract from PubMed 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 A 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. X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine.,Asgeirsson B, Markusson S, Hlynsdottir SS, Helland R, Hjorleifsson JG Biochem Biophys Rep. 2020 Oct 15;24:100830. doi: 10.1016/j.bbrep.2020.100830., eCollection 2020 Dec. PMID:33102813 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Alkaline phosphatase 3D structures

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