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Establishing the catalytic mechanism of human pancreatic α-amylase with QM/MM methods.
Pinto, Gaspar P; Brás, Natércia F; Perez, Marta A S; Fernandes, Pedro A; Russo, Nino; Ramos, Maria J; Toscano, Marirosa.
Afiliación
  • Pinto GP; Università della Calabria , Via Pietro Bucci, Arcavacata, Italia.
  • Brás NF; UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
  • Perez MA; UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
  • Fernandes PA; UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
  • Russo N; UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
  • Ramos MJ; Università della Calabria , Via Pietro Bucci, Arcavacata, Italia.
  • Toscano M; UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
J Chem Theory Comput ; 11(6): 2508-16, 2015 Jun 09.
Article en En | MEDLINE | ID: mdl-26575550
In this work, we studied the catalytic mechanism of human pancreatic α-amylase (HPA). Our goal was to determine the catalytic mechanism of HPA with atomic detail using computational methods. We demonstrated that the HPA catalytic mechanism consists of two steps, the first of which (glycosylation step) involves breaking the glycosidic bond to culminate in the formation of a covalent intermediate. The second (deglycosylation step) consists of the addition of a water molecule to release the enzyme/substrate covalent intermediate, completing the hydrolysis of the sugar. The active site was very open to the solvent. Our mechanism basically differs from the previously proposed mechanism by having two water molecules instead of only one near the active site that participate in the mechanism. We also demonstrate the relevant role of the three catalytic amino acids, two aspartate residues and a glutamate (D197, E233, and D300), during catalysis. It was also shown that the rate limiting step was glycosylation, and its activation energy was in agreement with experimental values obtained for HPA. The experimental activation energy was 14.4 kcal mol(-1), and the activation energy obtained computationally was 15.1 kcal mol(-1).
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Teoría Cuántica / Alfa-Amilasas Pancreáticas / Biocatálisis Límite: Humans Idioma: En Revista: J Chem Theory Comput Año: 2015 Tipo del documento: Article País de afiliación: Italia Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Teoría Cuántica / Alfa-Amilasas Pancreáticas / Biocatálisis Límite: Humans Idioma: En Revista: J Chem Theory Comput Año: 2015 Tipo del documento: Article País de afiliación: Italia Pais de publicación: Estados Unidos