Interactions defining the specificity between fungal xylanases and the xylanase-inhibiting protein XIP-I from wheat.
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 K(i) values for fungal xylanases ranged from 3.4 to 610 nM, 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 -1.38 kJ x mol(-1) x K(-1), leading to a predicted buried surface area of approx. 2200+/-500 A(2) at the complex interface. For this complex with A. niger xylanase (K(i)=320 nM 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 (K(i)=9 nM) 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 k(off) for the latter, with only a small difference in association rate k(on).
Flatman
Ruth
R
Institute of Food Research, Colney Lane, Norwich NR4 7UA, U.K.
McLauchlan
W Russell
WR
Juge
Nathalie
N
Furniss
Caroline
C
Berrin
Jean-Guy
JG
Hughes
Richard K
RK
Manzanares
Paloma
P
Ladbury
John E
JE
O'Brien
Ronan
R
Williamson
Gary
G
D001230:Aspergillus / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D007525:Isoelectric Focusing; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D014908:Triticum / Q000737:chemistry*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D010940:Plant Proteins; D014990:Xylans; C085118:arabinoxylan; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase