title: Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.
abstract: Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) 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 Mg(2)(+) 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, 15Å 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.
authors.affiliation: Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavik, Iceland. Electronic address: jgh4@hi.is.
authors.forename: Jens Guðmundur
authors.initials: JG
authors.lastname: Hjörleifsson
authors.affiliation: Department of Biochemistry, Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavik, Iceland. Electronic address: bjarni@hi.is.
authors.forename: Bjarni
authors.initials: B
authors.lastname: Ásgeirsson
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry*; D003080:Cold Temperature; D004795:Enzyme Stability; D017510:Protein Folding; D055503:Protein Multimerization; D014508:Urea / Q000494:pharmacology*; D014733:Vibrio / Q000201:enzymology
chemical_list: D014508:Urea; D000469:Alkaline Phosphatase
keywords: Alkaline phosphatase; Cold adaption; Dimer; Enzyme kinetics; Phosphorescence; Protein fluorescence