FEBS Letters | |
C‐terminal region contributes to muscle acylphosphatase three‐dimensional structure stabilisation | |
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
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
RO201912020302601ZK.pdf | 527KB | download |