Inhibition of lipase from rice (Oryza sativa) by diethyl-p-nitrophenyl phosphate

Abstract The inhibition of rice bran lipase (RBL) by
diethyl-p-nitrophenyl phosphate was studied with reference
to kinetics, nature of inhibition and also elucidate the effect
of the inhibitor on the structure—function of the enzyme.
Enzyme activity measurements shows that the inhibitor is
more effective at 0.050 mM concentration of diethyl-pnitrophenyl
phosphate and the activity is 50% at this level
of inhibitor concentration. The affinity of substrate for the
enzyme was observed by the increase in the velocity of the
reaction with increase in the substrate concentrations and
double reciprocal plot indicates that the inhibition followed
a competitive in nature and inhibition constant Ki is found
to be 0.016 mM at pH 7.0. The decrease in apparent
thermal denaturation temperature to 4 �C compared to
control indicates the destabilization of enzyme in the
presence of inhibitor. Fluorescence spectral measurements
suggests that pronounced quenching of fluorescence
intensity of RBL occurs at higher concentrations of diethylp-
nitrophenyl phosphate and ‘Ka’ value was found to be
2.4 · 104 M–1 with free energy change DGo—26 kJ/mol at
30 �C suggesting strong binding between the enzyme and
the inhibitor with microenvironmental changes occur at the
active site or in the neighbourhood of active site. The far
UV-CD data suggest that there is no significant changes in
the conformation of the enzyme as a result of binding of
diethyl-p-nitrophenyl phosphate. These results indicate that
diethyl-p-nitrophenyl phosphate is a inhibitor of RBL and
binds to the enzyme in brining about inhibition without any
structural alterations.