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Inhibition of α-amylase activity by Zn2+: insights from spectroscopy and molecular dynamics simulations.

S. Liao,N. Shen,Ge Liang,B. Lu,Zhi-long Lu,Li-Xin Peng,F. Zhou,Li-Qin Du,Yu-Tuo Wei,Guo-Ping Zhou,R. Huang

Published 2018 in Medicinal chemistry

ABSTRACT

BACKGROUND Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed. OBJECTIVE In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively). METHODS Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server (http://blast.ncbi.nlm.nih.gov) to find the most similar protein sequence to use as a template for homology modeling. The best template was selected based on the percentage of sequence similarity, the percentage of sequence identity, the percentage of coverage and the E-value. RESULTS According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and β-sheet, α-turn and random-coil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package. CONCLUSION This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

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