Effects induced by mono-divalent cations on protein's regions responsible for thermal adaptation in beta-glycosidase from Sulfolobus solfataricus

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus beta-glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1). an extra segment (83-124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2). a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the beta-glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg(2+). Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 degrees C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107-R245 and E109-R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A-C intermolecular interface of Sbetagly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474-K72 and D473-R402, with consequent partial dissociation of the tetrameric structure.