Investigation of the active site of the extracellular beta-D-xylosidase from Aspergillus carbonarius.
The catalytic amino acid residues of the extracellular beta-D-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Aspergillus carbonarius was investigated by the pH dependence of reaction kinetic parameters and chemical modifications of the enzyme. The pH dependence curves gave apparent pK values of 2.7 and 6.4 for the free enzyme, while pK value of 4.0 was obtained for the enzyme-substrate complex using p-nitrophenyl beta-D-xyloside as a substrate. These results suggested that a carboxylate group and a protonated group--presumably a histidine residue--took part in the binding of the substrate but only a carboxylate group was essential in the substrate cleavage. Carbodiimide- and Woodward's reagent K-mediated chemical modifications of the enzyme also supported that a carboxylate residue, located in the active center, was fundamental in the catalysis. The pH dependence of inactivation revealed the involvement of a group with pK value of 4.4, proving that a carboxylate residue relevant for hydrolysis was modified. During modification V(max) decreased to 10% of that of the unmodified enzyme and K(m) remained unchanged, supporting that the modified carboxylate group participated in the cleavage and not in the binding of the substrate. We synthesized and tested a new, potential affinity label, N-bromoacetyl-beta-d-xylopyranosylamine for beta-D-xylosidase. The A. carbonarius beta-D-xylosidase was irreversible inactivated by N-bromoacetyl-beta-D-xylopyranosylamine. The competitive inhibitor beta-D-xylopyranosyl azide protected the enzyme from inactivation proving that the inactivation took place in the active center. Kinetic analysis indicated that one molecule of reagent was necessary for inactivation of one molecule of the enzyme.
Kiss
Tünde
T
Institute of Biochemistry, University of Debrecen, H-4010 Debrecen, Hungary.
Erdei
Anikó
A
Kiss
László
L
D000345:Affinity Labels / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology*; D001665:Binding Sites; D002264:Carboxylic Acids / Q000737:chemistry; D007700:Kinetics; D009005:Monosaccharides / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D002264:Carboxylic Acids; D009005:Monosaccharides; C451059:n-bromoacetyl-xylopyranosylamine; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase