Inhibitory effects of 2-hydroxybenzaldehyde on the activity of phenoloxidase from Pieris rapae (Lepidoptera) larvae

Abstract

Phenoloxidase (PO) is a key enzyme in insect development, responsible for catalyzing the hydroxylation of tyrosine into O-diphenols and oxidation of O-diphenols into O-quinones. In the present study, the kinetic assay for PO from Pieris rapae (L.) larvae was performed in air-saturated solutions and its kinetic behavior in the oxidation of L-tyrosine (a monophenol) and L-DOPA (L-3, 4-dihydroxyphenylalanine, a diphenol) was studied. The inhibitory effects of 2-hydroxybenzaldehyde (2-HBD) on the monophenolase and diphenolase activities of PO were also studied. Results showed that 2-HBD inhibit both the monophenolase and diphenolase activities of PO. The lag period of L-tyrosine oxidation catalyzed by the enzyme was lengthened and the steady-state activity of the enzyme sharply decreased. The inhibitor was found to be noncompetitively reversible with a KI (KI = KIS) of 1.21 mmol/L and an estimated IC₅₀ of 8.08 ± 0.11 mmol/L for monophenolase and 4.14 ± 0.08 mmol/L for diphenolase activities. In the time-course of oxidation of L-DOPA catalyzed by the enzyme in the presence of different concentrations of 2-HBD, the rate decreased with increasing time until a straight line was reached. The microscopic rate constants for the reaction of 2-HBD with the enzyme were determined.