Investigation of the Active Site of the Extracellular β-d-Xylosidase from Aspergillus carbonarius

The catalytic amino acid residues of the extracellular β-d-xylosidase (β-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 β-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 Woodwardʹs 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 Vmax decreased to 10% of that of the unmodified enzyme and Km 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-β-d-xylopyranosylamine for β-d-xylosidase. The A. carbonarius β-d-xylosidase was irreversible inactivated by N-bromoacetyl-β-d-xylopyranosylamine. The competitive inhibitor β-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.