His374 of wheat endoxylanase inhibitor TAXI-I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases.
Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.
Fierens
Katleen
K
Katholieke Universiteit Leuven, Laboratory of Food Chemistry, Leuven, Belgium. katleen.fierens@biw.kuleuven.be
Gils
Ann
A
Sansen
Stefaan
S
Brijs
Kristof
K
Courtin
Christophe M
CM
Declerck
Paul J
PJ
De Ranter
Camiel J
CJ
Gebruers
Kurt
K
Rabijns
Anja
A
Robben
Johan
J
Campenhout
Steven
S
Volckaert
Guido
G
Delcour
Jan A
JA
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology; D001412:Bacillus subtilis / Q000201:enzymology; D002942:Circular Dichroism; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors* / Q000145:classification / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014242:Trichoderma / Q000201:enzymology; D014908:Triticum / Q000201:enzymology*
D010940:Plant Proteins; D011994:Recombinant Proteins; C422246:TAXI I protein, Triticum aestivum; D005973:Glutamine; D006639:Histidine; D043364:Endo-1,4-beta Xylanases; D008239:Lysine; D000409:Alanine