Article (Scientific journals)
Structural Determinants of Cold Adaptation and Stability in a Large Protein
D'Amico, Salvino; Gerday, Charles; Feller, Georges
2001In Journal of Biological Chemistry, 276 (28), p. 25791-6
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Abstract :
[en] The heat-labile alpha-amylase from an antarctic bacterium is the largest known protein that unfolds reversibly according to a two-state transition as shown by differential scanning calorimetry. Mutants of this enzyme were produced, carrying additional weak interactions found in thermostable alpha-amylases. It is shown that single amino acid side chain substitutions can significantly modify the melting point T(m), the calorimetric enthalpy Delta H(cal), the cooperativity and reversibility of unfolding, the thermal inactivation rate constant, and the kinetic parameters k(cat) and K(m). The correlation between thermal inactivation and unfolding reversibility displayed by the mutants also shows that stabilizing interactions increase the frequency of side reactions during refolding, leading to intramolecular mismatches or aggregations typical of large proteins. Although all mutations were located far from the active site, their overall trend is to decrease both k(cat) and K(m) by rigidifying the molecule and to protect mutants against thermal inactivation. The effects of these mutations indicate that the cold-adapted alpha-amylase has lost a large number of weak interactions during evolution to reach the required conformational plasticity for catalysis at low temperatures, thereby producing an enzyme close to the lowest stability allowing maintenance of the native conformation.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
D'Amico, Salvino ;  Université de Liège - ULiège > GIGA-Research
Gerday, Charles ;  Université de Liège - ULiège > Services généraux (Faculté des sciences) > Relations académiques et scientifiques (Sciences)
Feller, Georges ;  Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie
Language :
English
Title :
Structural Determinants of Cold Adaptation and Stability in a Large Protein
Publication date :
13 July 2001
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, United States - Maryland
Volume :
276
Issue :
28
Pages :
25791-6
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 26 January 2010

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