title: Establishing the catalytic mechanism of human pancreatic α-amylase with QM/MM methods.
abstract: 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).
authors.affiliation: Università della Calabria , Via Pietro Bucci, Arcavacata, Italia.|UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
authors.forename: Gaspar P
authors.initials: GP
authors.lastname: Pinto
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
authors.forename: Natércia F
authors.initials: NF
authors.lastname: Brás
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
authors.forename: Marta A S
authors.initials: MA
authors.lastname: Perez
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Fernandes
authors.affiliation: Università della Calabria , Via Pietro Bucci, Arcavacata, Italia.
authors.forename: Nino
authors.initials: N
authors.lastname: Russo
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto , Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Ramos
authors.affiliation: Università della Calabria , Via Pietro Bucci, Arcavacata, Italia.
authors.forename: Marirosa
authors.initials: M
authors.lastname: Toscano
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D055292:Pancreatic alpha-Amylases / Q000737:chemistry* / Q000378:metabolism*; D011789:Quantum Theory
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid; D055292:Pancreatic alpha-Amylases