Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.
Biochem Biophys Res Commun
; 721: 150109, 2024 Aug 20.
Article
em En
| MEDLINE
| ID: mdl-38762932
ABSTRACT
Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.
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Base de dados:
MEDLINE
Assunto principal:
Estabilidade Enzimática
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Cálcio
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Endopeptidase K
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Estabilidade Proteica
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Simulação de Dinâmica Molecular
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article