Abstract
The tryptophanyl residues of the Aspergillus niger glucoamylase G1 and G2 (EC 3.2.1.3) were oxidized by N-bromosuccinimide in both the presence and the absence of substrates and inhibitors of the enzyme. In the absence of protective ligands, 8 of 19 and 6 of 15 tryptophanyl residues in G1 and G2, respectively, were susceptible to modification with concomitant inactivation of the enzyme. The binding of acarbose, a potent inhibitor, prior to oxidation protected 2 tryptophanyl residues (Wa and Wb) in both G1 and G2 from the modification. After dissociation of acarbose-enzyme complexes with the aid of Tris, these derivatives retained about 80% of the initial enzymic activity. Further oxidation subsequently modified these 2 tryptophanyl residues resulting in total loss of activity. The substrates, maltose, maltotriose or soluble starch and the inhibitors, gluconolactone, maltitol or deoxynojirimycin protected one tryptophanyl residue (Wb) in both G1 and G2 from oxidation but did not prevent the inactivation.
Characterization of the oxidized enzyme derivatives by difference UV absorption and by fluorescence spectroscopy indicated that 2 residues, Wa and Wb, are essential in the mechanism of glucoamylase action. One residue, Wb, is apparently involved in the binding of substrate, while a second, Wa, is an integral part of a catalytically capable active center.
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Abbreviations
- DPCC:
-
diphenylcarbamyl chloride
- G1 and G2:
-
designate the larger and smaller forms, respectively, of glucoamylase from A, niger (29)
- NBS:
-
N-bromosuccinimide
- nWox :
-
number (n) of oxidized tryptophanyl residues in isolated derivatives
- Tris:
-
2-amino-2(hydroxymethyl)-1,3-propandiol
- Wa and Wb :
-
ligand protected tryptophanyl residues
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Clarke, A.J., Svensson, B. The role of tryptophanyl residues in the function of Aspergillus niger glucoamylase G1 and G2. Carlsberg Res. Commun. 49, 111–122 (1984). https://doi.org/10.1007/BF02913970
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DOI: https://doi.org/10.1007/BF02913970