Biochemical Characterization of a Novel Aspartic Protease from Plutella xylostella, Diamondback moth (Lepidoptera: Plutellidae) Midgut: Inactivation by an Aspartic Protease Inhibitor

Abstract

An aspartic protease from Plutella xylostella (PxAP) was purified to homogeneity by ammonium sulphate precipitation; gel filtration and affinity chromatography with 110 fold purification. The enzyme exhibited a Mr. of 46.1kDa on SDS-PAGE with an optimum pH and temperature of 3.5 and 37°C respectively. The enzyme hydrolyzed hemoglobin with a K M value of 538 µM . The enzyme properties are consistent with those of aspartic proteases as it was inhibited by pepstatin and also by an aspartic protease inhibitor, API. The inhibition of PxAP by API followed a hyperbolic pattern with increasing concentrations of inhibitor with an IC 50 value of 44μM. The inhibition constant K i determined by Lineweaver-Burk and Dixon plot was found to be 59μM and 55μM respectively. The Kinetic studies on the PxAP-API interactions reveal a slow-tight binding competitive mechanism of inhibition with the overall inhibition constant K i *= 3.7 ± 0.3nM . The inhibition progress curves are time-dependent and consistent with slow-tight binding inhibition: E+I ⇄ ( k 3 , k 4 )EI ⇄ ( k 5 , k 6 )EI*. The rate constant k 5 =6.64 ± 0.7 ´ 10 -2 s -1 reveals a fast isomerization of enzyme-inhibitor complex [EI] and a very slow dissociation of EI* complex, which is proved by rate constant k 6 =5.67 ± 0.9 ´ 10 -6 s -1 . The Rate constants from the intrinsic tryptophanyl fluorescence data are in agreement with those obtained from the kinetic analysis, therefore the induced conformational changes in the enzyme were correlated to the isomerization of EI to EI*.