|Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted α-amylase|D'Amico, S.; Gerday, C.; Feller, G. (2002). Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted α-amylase. J. Biol. Chem. 277(48): 46110-46115. dx.doi.org/10.1074/jbc.M207253200
In: Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology: Baltimore, etc.. ISSN 0021-9258, more
Chloride-dependent α-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile α-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (ΔΔG = 1.87 kcal/mol at 20 °C) when compared with the wild-type enzyme, although the melting point (T m) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated α-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 °C as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution.