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Chemical modification of barley malt α-amylase 2: Involvement of tryptophan and tyrosine residues in enzyme activity

1986; Springer Nature; Volume: 51; Issue: 5 Linguagem: Inglês

10.1007/bf02907163

0105-1938

Richard M. Gibson, Birte Svensson,

Biochemical and Molecular Research

Barley malt α-amylase 2 has been purified on a large scale and the resulting enzyme preparation used in various chemical modification experiments. Results from tryptophan and tyrosine specific modifying reagents, as well as difference and fluorescence spectroscopy techniques, which studied the interaction of a variety of ligands, indicate that tryptophanyl and tyrosyl residues are involved in enzyme activity. Of the 16 tryptophanyl residues present in the enzyme, 4 could be modified using dimethyl (2-hydroxy-5-nitrobenzyl)sulphonium bromide. This caused inactivation of the enzyme. Addition of the inhibitor aplanin, a pseudo-maltooligosaccharide, decreased the loss of enzyme activity and protected 2 tryptophanyl residues from modification. Aplanin also protected the enzyme from inactivation by tetranitromethane, a tyrosine specific reagent. β-Cycloheptaamylose appears to interact with a tryptophanyl residue not essential for enzyme activity, suggesting that this residue may be located in a second ligand binding site on the enzyme. This is in agreement with previous predictions of a "surface" site on barley malt α-amylase.