Establishing the Catalytic Mechanism of Human Pancreatic alpha-Amylase with QM/MM Methods

• Type: Article in International Scientific Journal
• Date: 2015

Authors

Gaspar P Pinto (Author) Other; Natercia F Bras (Author) Other; Marta A S Perez (Author) Other; Pedro A Fernandes (Author) FCUP; Nino Russo (Author) Other; Maria J Ramos (Author) FCUP; Marirosa Toscano (Author) Other

Journal

Title: Journal of Chemical Theory and Computation

Vol. 11

Pages: 2508-2516

ISSN: 1549-9618

Publisher: American Chemical Society

Indexing

ISI Web of Knowledge - 29 Citations

Scopus - 28 Citations

Scientific classification

FOS: Natural sciences > Chemical sciences

Other information

Authenticus ID: P-00G-BRW

DOI: 10.1021/acs.jctc.5b00222

Abstract (EN): In this work, we studied the catalytic mechanism of human pancreatic alpha-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).

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 9