Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase Bjarni Ásgeirsson ⁎ , Björn Vidar Adalbjörnsson, Gudjón Andri Gylfason Department of Biochemistry, Science Institute, University of Iceland, Dunhaga 3, IS107 Reykjavík, Iceland Received 9 September 2006; received in revised form 24 March 2007; accepted 26 March 2007 Available online 5 April 2007 Abstract Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 Å radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild- type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k cat ) and a general decrease in K m values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP. © 2007 Elsevier B.V. All rights reserved. Keywords: Cold-adaptation; Site-directed mutagenesis; Catalytic efficiency; Cysteine; Disulfide bond 1. Introduction The Earth's biosphere is dominated by cold habitats, and therefore, cold-adaptation of enzymes is the norm rather than the exception. Most such enzymes meet the challenge to drive reactions forward in environments of low thermal energy by attaining the required dynamic catalytic mobility [1–4]. As the temperature increases, these enzymes commonly suffer tem- perature denaturation, since evolutionary pressure has not directed residue selection to deal with temperature stability. Several factors contribute to protein stability. In addition to hydrophobic interactions, non-covalent interactions such as hydrogen bonds, van der Waals interactions, and ion-pair networks (salt-bridges) can provide all of the stabilizing energy needed. Disulfide bonds are the covalent bonds that proteins can further utilize for stability, and they do contribute significantly to stability of several proteins [5–7]. Disulfide bonds are only found in some extracellular proteins due to the unfavorable intracellular redox state [5,8]. They give proteins extra stability and may promote folding into the catalytically active con- formation. Furthermore, the function of some secreted soluble proteins and cell-surface receptors is controlled by cleavage of disulfide bonds by specific catalysts or facilitators [9]. The stabilizing effect of disulfide bonds is commonly linked to a reduction in main-chain entropy of the unfolded state [6,10]. Interactions of the sulfur atoms and aromatic groups show high degree of preservation and may provide extra overall stability as well as set the orientation of aromatic rings [11]. The stabilizing energy provided by a single disulfide bond is 2–5 kcal/mol [12,13]. Although the introduction of new disulfide bridges by genetic engineering often leads to the expected stabilization, many mutants show no effect, or even destabilization, when compared with the wild-type enzyme [10,14–16]. Possible reasons for detrimental effects include strain caused by the introduction of a disulfide bond because the required stereo- chemistry is not exact, or because substituting a residues with cysteine loses some favorable interactions, or causes steric contact strain [17,18]. Cold-adaptation of enzymes is believed to involve less internal adhesion in the protein's structure in order to allow more dynamic movement within the active site. It is not always Biochimica et Biophysica Acta 1774 (2007) 679 – 687 www.elsevier.com/locate/bbapap ⁎ Corresponding author. Tel.: +354 525 4800; fax: +354 552 8911. E-mail address: bjarni@raunvis.hi.is (B. Ásgeirsson). 1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2007.03.016