Chloride-dependent alpha-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 alpha-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 (DeltaDeltaG = 1.87 kcal/mol at 20 degrees 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 alpha-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degrees 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. Study holds ProTherm entries: 17640, 17641, 17642, 17643, 17644, 17645, 17646, 17647, 17648 Extra Details: Chloride-dependent; cold-active; catalytic parameters; compact structure;,enthalpy-entropy compensation effect
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:49 p.m.
|Number of data points||17|
|Proteins||Alpha-amylase ; Pancreatic alpha-amylase|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: dG ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence TPTTFVHLFEWNWQDVAQECEQYLGPKGYAAVQVSPPNEHITGSQWWTRYQPVSYELQSRGGNRAQFIDMVNRCSAAGVDIYVDTLINHMAAGSGTGTAGNSFGNKSFPIYSPQDFHESCTINNSDYGNDRYRVQNCELVGLADLDTASNYVQNTIAAYINDLQAIGVKGFRFDASKHVAASDIQSLMAKVNGSPVVFQEVIDQGGEAVGASEYLSTGLVTEFKYSTELGNTFRNGSLAWLSNFGEGWGFMPSSSAVVFVDNHDNQRGHGGAGNVITFEDGRLYDLANVFMLAYPYGYPKVMSSYDFHGDTDAGGPNVPVHNNGNLECFASNWKCEHRWSYIAGGVDFRNNTADNWAVTNWWDNTNNQISFGRGSSGHMAINKEDSTLTATVQTDMASGQYCNVLKGELSADAKSCSGEVITVNSDGTINLNIGAWDAMAIHKNAKLNTSSAS ; Mutations for sequence QYAPQTQSGRTSIVHLFEWRWVDIALECERYLGPKGFGGVQVSPPNENVVVTNPSRPWWERYQPVSYKLCTRSGNENEFRDMVTRCNNVGVRIYVDAVINHMCGSGAAAGTGTTCGSYCNPGSREFPAVPYSAWDFNDGKCKTASGGIESYNDPYQVRDCQLVGLLDLALEKDYVRSMIADYLNKLIDIGVAGFRIDASKHMWPGDIKAVLDKLHNLNTNWFPAGSRPFIFQEVIDLGGEAIKSSEYFGNGRVTEFKYGAKLGTVVRKWSGEKMSYLKNWGEGWGFMPSDRALVFVDNHDNQRGHGAGGSSILTFWDARLYKVAVGFMLAHPYGFTRVMSSYRWARNFVNGEDVNDWIGPPNNNGVIKEVTINADTTCGNDWVCEHRWREIRNMVWFRNVVDGEPFANWWDNGSNQVAFGRGNRGFIVFNNDDWQLSSTLQTGLPGGTYCDVISGDKVGNSCTGIKVYVSSDGTAQFSISNSAEDPFIAIHAESKL|