Mutational analysis of divalent metal ion binding in the active site of class II α-mannosidase from Sulfolobus solfataricus.
Biochemistry
; 54(11): 2032-9, 2015 Mar 24.
Article
en En
| MEDLINE
| ID: mdl-25751413
Mutational analysis of Sulfolobus solfataricus class II α-mannosidase was focused on side chains that interact with the hydroxyls of the -1 mannosyl of the substrate (Asp-534) or form ligands to the active site divalent metal ion (His-228 and His-533) judged from crystal structures of homologous enzymes. D534A and D534N appeared to be completely inactive. When compared to the wild-type enzyme, the mutant enzymes in general showed only small changes in K(M) for the substrate, p-nitrophenyl-α-mannoside, but elevated activation constants, K(A), for the divalent metal ion (Co²âº, Zn²âº, Mn²âº, or Cd²âº). Some mutant enzyme forms displayed an altered preference for the metal ion compared to that of the wild type-enzyme. Furthermore, the H228Q, H533E, and H533Q enzymes were inhibited at increasing Zn²âº concentrations. The catalytic rate was reduced for all enzymes compared to that of the wild-type enzyme, although less dramatically with some activating metal ions. No major differences in the pH dependence between wild-type and mutant enzymes were found in the presence of different metal ions. The pH optimum was 5, but enzyme instability was observed at pH <4.5; therefore, only the basic limb of the bell-shaped pH profile was analyzed.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Cationes Bivalentes
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Proteínas Arqueales
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Alfa-Manosidasa
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Sulfolobus solfataricus
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Proteínas Mutantes
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Metales
Idioma:
En
Revista:
Biochemistry
Año:
2015
Tipo del documento:
Article
País de afiliación:
Dinamarca
Pais de publicación:
Estados Unidos