C‐terminal region contributes to muscle acylphosphatase three‐dimensional structure stabilisation

FEBS Letters

Chiti, Fabrizio¹  Stefani, Massimo¹  Magherini, Francesca¹  Raugei, giovanni¹  Taddei, Niccolò¹  Bucciantini, Monica¹  Ramponi, Giampietro¹ 

[1] Department of Biochemical Sciences, University of Florence, V.le Morgagni 50, 50134 Florence, Italy

关键词: Acylphosphatase;    deletion mutant;    Acylphosphatase;    insertion mutant;    Acylphosphatase;    stability;    Acylphosphatase;    1H NMR spectrum;    Δ+2;    acylphosphatase mutant containing a Ser-Ala extension at the C-terminus;    Δ+4;    acylphosphatase mutant containing a Ser-Ala-Ser-Ala extension at the C-terminus;    Δ−2;    acylphosphatase mutant lacking the C-terminal dipeptide;    Δ−3;    acylphosphatase mutant lacking the C-terminal tripeptide;    IPTG;    isopropyl thiogalactoside;    GST;    glutathione S-transferase;    NMR;    nuclear magnetic resonance;   

DOI  :  10.1016/0014-5793(96)00292-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Ser-Ala and Ser-Ala-Ser-Ala C-terminus elongated (Δ+2 and Δ+4, respectively) and two C-terminus deleted (Δ−2 and Δ−3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C-terminal region. The kinetic analysis of these mutants shows that the removal of two or three C-terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild-type enzyme, respectively; instead, the elongation of the C-terminus does not significantly change the enzyme behaviour. ¹H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native-like fold though they appear less stable, particularly Δ−2 and Δ−3 mutants, as compared to the wild-type enzyme. Such destabilisation of the C-terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C-terminal region in the stabilisation of the three-dimensional structure of acylphosphatase, particularly at the active-site level. Moreover, a participation of the C-terminal carboxyl group to the catalytic mechanism can be excluded.

【 授权许可】

Unknown