The inhibitory effect of citrus flavonoids naringenin and hesperetin against purine nucleoside phosphorylase: Spectroscopic, atomic force microscopy and molecular modeling studies

In this work, the inhibitory effect of two citrus flavonoids naringenin and hesperetin on human purine nucleoside phosphorylase (hPNP) and their binding mechanism were evaluated. Results from enzymatic kinetics revealed that naringenin and hesperetin reversibly inhibited hPNP via a mixed-type manner with IC 50 values of 4.83 × 10 −4  M and 5.32 × 10 −4  M, respectively. Analysis of molecular modeling revealed that both naringenin and hesperetin bound directly into the active site by generating multiple forces including hydrogen bonding , π–π and π-Alkyl interactions with His64, Glu201, Ser220, His257, Phe200 and Val217 residues of hPNP, which caused the inhibition of hPNP activity. Moreover, conformational analysis by three-dimension fluorescence, circular dichroism and atomic force microscopy revealed that the binding of naringenin and hesperetin to hPNP induced changes in the microenvironment, secondary structure and morphology of hPNP. These results suggested that occupying the active site and enzymatic conformational perturbation induced by naringenin and hesperetin are the main reasons for reducing the inhibition of hPNP activity, which would be helpful in understanding the inhibitory mechanism of naringenin and hesperetin against hPNP.