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
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 to find
the most similar protein sequence to use as a template for homology modeling. 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.
Results: According to circular dichroism experiments, upon Zn2+ binding, the protein secondary
structure changed obviously, with the α-helix content decreasing and β-sheet, β-turn and randomcoil
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.
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.