The Contribution of Acidic Residues to the Conformational Stability of Common-Type Acylphosphatase

Abstract

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 All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase.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, ⌬G(H 2 O), 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.

Folding and Aggregation Are Selectively Influenced by the Conformational Preferences of the α-Helices of Muscle Acylphosphatase

“…It has been previously suggested that enzymatic activity can be used as a probe for the assessment of the native-like structure of an AcP protein variant, provided that the mutated residue does not participate to the catalytic mechanism and/or does not contribute to the formation of the active site (26). The activity measurements performed here clearly show that all these AcP variants maintain a native-like structural architecture.…”

Structural and Thermodynamic Investigations on the Aggregation and Folding of Acylphosphatase by Molecular Dynamics Simulations and Solvation Free Energy Analysis

“…The protein engineering method to study the mutation effects on AcP unfolding and aggregation has been actively applied to describe amyloid fibril formation as well as protein structure–function relationships. ,,− Dobson and co-workers have systematically reported the effects of single point mutations of AcP and found that V9A, E29D, A30G, S87T, and S92T mutations showed increased aggregation rate, whereas V20A, G34A, V39A, L89A, Y91Q, and Y98Q mutations displayed the opposite result. ,, Among those mutations, A30G accelerates and Y91Q decelerates the aggregation rate most distinctively. ,, On the other hand, both of those mutations contribute to slow down the folding rate of AcP with respect to the wild type. , Despite the extensive mutation experiments on AcP protein, the molecular origin and the structural motifs to promote the aggregation and folding rate changes have not been rationalized at the atomic-level due to the absence of the AcP mutant structures. Flöck et al have previously performed the molecular dynamics (MD) simulations for AcP monomer of its wild type under 25% (v/v) trifluoroethanol (TFE)/water solvent and found the enhancement of aggregation propensity mainly due to the TFE and β-propensity of the α1-helix (residues 22–33) .…”

The intrachain disulfide bridge is responsible of the unusual stability properties of novel acylphosphatase from Escherichia coli

“…Hu-CT [21] 841 ± 156 5.0-6.0 0.15 ± 0.01 0.59 ± 0.04 5610 ± 670 Hu-MT [22] 1224 ± 120 4.8-5.8 0.34 ± 0.03 0.75 ± 0.08 3420 ± 500 AcPDro [7] 35 ± 3 5. Table 2.…”