Interactions defining the specificity between fungal xylanases and the xylanase-inhibiting protein XIP-I from wheat

Abstract

We previously reported on the xylanase-inhibiting protein I (XIP-I) from wheat [McLauchlan, Garcia-Conesa, Williamson, Roza, Ravestein and Maat (1999), Biochem. J. 338, 441–446]. In the present study, we show that XIP-I inhibits family-10 and −11 fungal xylanases. The Ki values for fungal xylanases ranged from 3.4 to 610nM, but bacterial family-10 and −11 xylanases were not inhibited. Unlike many glycosidase inhibitors, XIP-I was not a slow-binding inhibitor of the Aspergillus niger xylanase. Isothermal titration calorimetry of the XIP-I—A. niger xylanase complex showed the formation of a stoichiometric (1:1) complex with a heat capacity change of −1.38kJ·mol−1·K−1, leading to a predicted buried surface area of approx. 2200±500Å2 at the complex interface. For this complex with A. niger xylanase (Ki = 320nM at pH 5.5), titration curves indicated that an observable interaction occurred at pH 4–7, and this was consistent with the pH profile of inhibition of activity. In contrast, the stronger complex between A. nidulans xylanase and XIP-I (Ki = 9nM) led to an observable interaction across the entire pH range tested (3–9). Using surface plasmon resonance, we show that the differences in the binding affinity of XIP-I for A. niger and A. nidulans xylanase are due to a 200-fold lower dissociation rate koff for the latter, with only a small difference in association rate kon.