Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations

• Published in: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
• Main Authors: Hjörleifsson, Jens G, Ásgeirsson, Bjarni
• Other Authors: Raunvísindadeild (HÍ), Faculty of Physical Sciences (UI), Raunvísindastofnun (HÍ), Science Institute (UI), Verkfræði- og náttúruvísindasvið (HÍ), School of Engineering and Natural Sciences (UI), Háskóli Íslands, University of Iceland
• Format: Article in Journal/Newspaper
• Language: English
• Published: Elsevier BV 2016
• Subjects: Alkaline phosphatase; Dimer; Enzyme kinetics; Protein fluorescence; Phosphorescence; Cold adaption; Fosfatasar; Lífefnafræði; Fosfór; Sameindir; Ensím; Iceland
• Online Access: https://hdl.handle.net/20.500.11815/2009; https://doi.org/10.1016/j.bbapap.2016.03.016

Post-print (lokagerð höfundar) Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn2+ and Mg2+ are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg2+ in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp → Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15 Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. Financial support from the Icelandic Research Fund (project 141619-051) and the Science Institute of the University of Iceland is gratefully acknowledged. The authors also extend their gratitude to Tinna Pálmadóttir for performing the experiment of Fig. 2B. Peer reviewed