The contribution of acidic residues to the conformational stability of common-type acylphosphatase

Common-type acylphosphatase is a small cytosolic enzyme whose catalytic pro perties 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 All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosph atase. 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 conform ational stability, Delta G(H2O), and specific activity values as compared t o the wild-type enzyme. By contrast, six mutants show a significant reducti on of conformational stability and two mutants are more stable than the wil d-type protein. Although none of the mutated acidic residues is directly in volved in the catalytic mechanism of the enzyme, owl 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 foun d a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measure ments can provide valuable indications on the conformational stability of a cylphosphatase mutants, provided the mutated residue lies far apart from th e active site, Moreover, our results indicate that the distortion of hydrog en bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein. (C) 1999 Acad emic Press.