Paoli P, Taddei N, Fiaschi T, Veggi D, Camici G, Manao G, Raugei G, Chiti F, Ramponi G
Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, Firenze, 50134, Italy.
Arch Biochem Biophys. 1999 Mar 15;363(2):349-55. doi: 10.1006/abbi.1998.1097.
Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, DeltaG(H2O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.
