Reversible inactivation of alkaline phosphatase from Atlantic cod (Gadus morhua) in urea Bjarni Ásgeirsson ⁎ , Katrín Guðjónsdóttir Department of Biochemistry, Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland Received 30 June 2005; received in revised form 9 December 2005; accepted 14 December 2005 Available online 10 January 2006 Abstract Alkaline phosphatase (AP) from Atlantic cod (Gadus morhua) is a zinc and magnesium containing homodimer that requires the oligomeric state for activity. Its kinetic properties are indicative of cold-adaptation. Here, the effect of urea on the structural stability was studied in order to correlate the activity with metal content, the microenvironment around tryptophan residues, and events at the subunit interface. At the lowest concentrations of urea, the first detected alteration in properties was an increase in the activity of the enzyme. This was followed by inactivation, and the release of half of the zinc content when the amount of urea reached levels of 2 M. Intrinsic tryptophan fluorescence and circular dichroism ellipticity changed in the range 2.5 to 8 M urea, signaling dimer dissociation, followed by one major monomer unfolding transition at 6–8 M urea as indicated by ANS fluorescence and KI fluorescence quenching. Gibbs free energy was estimated by the linear extrapolation method using a three-state model as 8.6 kcal/mol for dimer stability and 11.6 kcal/mol for monomer unfolding giving a total of 31.8 kcal/mol. Dimer association had a very small ionic contribution. Dimers were stable in relatively high concentration of urea, whereas the immediate vicinity around the active site was vulnerable to low concentrations of urea. Thus, inactivation did not coincide with dimer dissociation, suggesting that the active site is the most dynamic part of the molecule and closest related to cold-adaptation of its enzymatic activity. © 2005 Elsevier B.V. All rights reserved. Keywords: Cold-adaptation; Psychrophilic; Dimer; Metalloenzyme; Folding; Stability 1. Introduction Cold-adapted enzymes have evolved to function well at low temperatures, but at the same time, their temperature stability is lower than in homologous enzymes from warmer environments [1–3]. There has been some debate as to whether this global instability is necessary to ensure sufficient flexibility for catalytic movement by reducing weak non-covalent interactions in the structure, or due to lack of evolutionary pressure in the cold [4,5]. There is ample experimental evidence that enzyme activity requires some minimal mobility in active-sites, although it is not fully understood how the global dynamic motions of protein molecules contribute to their catalytic mechanisms [6]. Thus, the global stability of an enzyme is not necessarily an accurate indication of the flexibility at its active site, and inactivation of enzymes often precedes measurable global conformation changes for this reason. For the catalytic efficiencies of enzymes from psychrophilic organisms to be maintained at low temperatures, flexibility at the active site must be comparable to heat-adapted homologues [1–3]. Studies on cold-active enzymes have revealed that mutations can affect the mobility of specific parts that are involved in rate- determining catalytic conformational changes. Examples in- clude lactate dehydrogenase [7], chitobiase from a psychro- philic Antarctic bacterium [8], and uracil DNA glycosylase [9]. Dimerization is the most common oligomeric form of proteins, bringing advantages in terms of stability and activity [10,11]. Alkaline phosphatase is a good example where monomers are inactive and the dimer intersubunit surface shapes the functional form of the catalytic site. Thus, dimerization may determine the catalytic efficiency of a particular enzyme through regulation of conformational Biochimica et Biophysica Acta 1764 (2006) 190 – 198 http://www.elsevier.com/locate/bba Abbreviations: AP, Alkaline phosphatase (EC 3.1.3.1); ANS, 8-Anilino-1- naphtalene sulfonic acid; GdmCl, Guanidine hydrochloride; PAR, 4-(2- pyridylazo)resorcinol ⁎ Corresponding author. Tel.: +354 525 48 00; fax: +354 552 89 11. E-mail address: bjarni@raunvis.hi.is (B. Ásgeirsson). 1570-9639/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2005.12.015