The interaction of the elastase inhibitor, eglin c, with insect digestive endopeptidases: Effect of pH on the dissociation constants

Abstract

Midgut endopeptidases with N-succinyl-ala-ala-pro-leu p-nitroanilide-hydrolysing activities were partially purified from two species of lepidopteran larvae. The two enzymes from Cydia pomonella and the three enzymes from Heliocoverpa punctigera all showed a 2–5-fold increase in maximum rates and in Michaelis constants for the substrate between pH 7.6 and 10.5. Values for k cat at pH 10.5 varied from 5.3 to 21 s −1; and for K m between 0.76 and 4.05 mM. The chymotrypsin/elastase inhibitor, Eglin c, was not displaced by substrate from these enzymes under assay conditions. The K i values decreased 2–5-fold over the same pH range, being 1.2–2.5 × 10 −6 M for C. pomonella endopeptidases and 1.3–5.5 × 10 −8 M for H. punctigera endopeptidases at pH 10.5. The relative insensitivity of these parameters contrasts with those of bovine trypsin over a similar pH range below its pH optimum of pH 8.0.