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 acarbose 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, tryptie fragments of the two G2 derivatives were isolated. The peptide fragment from the inactive G2 der/vative containing the additional oxindolealaniae residue was Tocated in HPLC chromatograms by UV-absorption measurements. Normal and second derivative UV-spectra, amino acid composition and NH_,-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)).

Side chain reactivities of glucoamylase G2 from aspergillus niger evaluated by group-specific chemical modifications

“…For nitration (37) G2 (5 rag. ml" in 0.2 M sodium borate pH 8.2) was incubated with tetranitromethane (25 mM) at room temperature for 2 h, the derivative was purified by gel filtration followed by affinity chromatography (9). All these reactions took place in the dark.…”

“…Enzymatically active N-bromosuccinimide oxidized or carbodiimide modified G2 were prepared in the presence of acarbose as earlier described (8,9,42,46).…”

“…The acarbose concentration was 0.5 mg. ml-'. Acarbose and excess reagents were removed by dialysis (9) and the remaining histidine content measured by amino acid analysis. Enzyme activity was assayed using maltose.…”

“…The two forms are equally active towards soluble substrates, but only G 1 is able to degrade raw starch (47). A number of tryptophanyl residues and carboxyl groups involved in substrate binding and catalysis have been identified by chemical modification and sequencing (8,9,42,46). The related enzymes, a-amylase and ct-glucosidase, m adchtlon possess critical histidyl residues (2, 6,11,13,14,19,23,38), but modification of glucoamylase by diethyl pyrocarbonate ( 18,31,35) has only been found to decrease the affinity for soluble starch (18).…”

“…The related enzymes, a-amylase and ct-glucosidase, m adchtlon possess critical histidyl residues (2, 6,11,13,14,19,23,38), but modification of glucoamylase by diethyl pyrocarbonate ( 18,31,35) has only been found to decrease the affinity for soluble starch (18). In the light of the structural and functional similarities recognized recently for different starch-degrading enzymes (9,20,41), the role of histidyl and other side chains was reinvestigated in A. niger glucoamylase. The observed poor reactivity of groups normally exposed in native proteins motivated a characterization of the ionization behaviour of glucoamylase G2 by potentiometric and spectrophotometric titration.…”