Identification of an essential tryptophanyl residue in the primary structure of glucoamylase G2 from aspergillus niger

Abstract

Enzymatically active, N-bromosuccinimide oxidized glucoamylase G2 (EC 3.2 1.3) was prepared in the presence of the inhibitor nearbose and inactive, oxidized G2 with capacity to bind substrate was prepared in the presence of maltose. Four out of 15 tryptophanyl residues were oxidized in the first derivative and five in the latter. In order to identify this fifth and essential residue, tryptic fragments of the two G2 derivatives were isolated. The peptide fragment from the inactive G2 derivative containing the additional oxindolealanine residue was located in HPLC chromatograms by UV-absorption measurements. Normal and second derivative UV-spectra, amino acid composition and NH2-terminal sequence of the isolated fragment led to identification of Trp(120) as a residue essential for activity. A short homologous amino acid sequence precedes both this Trp(120) and the Trp(83) which is involved in substrate binding in Taka-amylase A (J. Biochem. 95. 697–702 (1984)).