How Stable is an Enzyme from a Thermophilic Organism? Denaturation Studies with the Esterase from Pyrococcus Furiosus - The Role of Charge-Charge Interactions

Abstract

We have expressed the gene of the hyperthermophilic esterase (Pf2001Δ60) from Pyrococcus furiosus. This esterase showed to be active after boiling and has a half-life of 120 min at 75 °C. We decided to study the unfolding of this enzyme by fluorescence spectroscopy induced by urea, guanidinium hydrochloride (GndHCl) and high hydrostatic pressure (HHP). Ours results pointed out that urea is about three times more efficient than GndHCl in promoting structural perturbations in PF2001Δ60 ([D1/2]=7.1 and 2.3 M, respectively), suggesting that ion pairs are important stabilizing factors on its structure. There was almost no change in the tryptophan center of mass of PF2001Δ60 (342 nm) under HHP up to 3.1 kbar, even by staying 120 min under 2,500 bar. Interestingly, the combination of HHP with a subdenaturating concentration of urea (1 M) displaced the center of mass by ≈ 7 nm after 40 min at 2,500 bar. Since HHP enhances the electrostriction, this result reinforces the crucial contribution of salt bridges in esterase's stability. The binding of the bis(8-anilinonaphthalene-1-sulfonate) to PF2001Δ60 was increased by 2.5- and 3-fold after treatment with 2 M GndHCl or under HHP combined with urea, suggesting that these treatments convert the enzyme into a partially folded intermediate with exposed hydrophobic regions. Altogether, ours results may be an indication that the optimization of charge-charge interactions on the protein surface is a key factor for its stability. To our knowledge, this is the first time that HHP is used to access the ion pair contributions to the stability of a hyperthermophilic esterase.