| Research Abstract |
Thaumatin, a sweet protein that contains no cysteine residues and eight intramolecular disulfide bonds, aggregates upon heating at pH 7.0 above 70℃ and its sweetness thereby disappears. The aggregate can be solubilized by heating in the presence of both thiol reducjng reagent and SDS. This molecular aggregation depended on the protein concentration during heating and was suppressed by the addition of N-ethylmaleimide or iodoacetamide, indicating a thiol-catalyzed disulfide interchange reaction between heat-denatured molecules. An amino acid analysis of the aggregates suggested that the cysteine and lysine residues were reduced, and the formation of a cysteine residue and a lysinoalanine residue was confirmed. The reduction and formation of these residues stoichiometrically satisfied the B-elimination of a cystine residue. The disulfide interchange reaction was catalyzed by cysteine ; I.e., a free sulfhydryl residue was formed via B-elimination of a disulfide bond. Intermolecular disulf
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ide bond were probably formed between thaumatin molecules upon heating at pH 7.0, which led to the aggregation of thaumatin molecules.
Thaumatin I is the sweet tasting protein isolated from the arils of a plant native to tropical West Africa. However, despite its strong sweetness, the structural basis for its sweetness is still unknown. We identified the functionally important lysine residues by using chemical modification study. Pyridoxal 5'-phosphate (PLP) was used to selectively modify lysine residues on thaumatin I. PLP was incubated with thaumatin I at pH 7.0, and the schiff base was reduced with sodium borohydride. Five modified thaumatin I, all of which were incorporated one PLP per thaumatin I molecule, were purified and its modified lysine residues were identified. Lys78, kys97, Lys106, Lys137, and Lys187 were modified, and any of these modification did not affect the conformation of thaumatin I as inferred from the measurement of circular dichroism spectra. Modified thaumatin I showed reduced sweetness, 67-85% loss. The modified thaumatin I was treated with phosphatase to remove phosphate group of attached PLP. Removal from Lys78-, Lys97-, Lys137-, and Lys187-modified-thaumatin I restored intense sweetness comparable to that of native thaumatin I, whereas removal from Lys106-modified-thaumatin I failed to restore sweetness. These results reveal that these five lysine residues, Lys78, Lys97, Lys106, Lys137, and Lys187 are functionally important. Our results also suggest, although the roles of Lys106 is obscure, that positive charge of Lys78, Lys97, Lys137, and Lys187 is participated in electrostatic interaction with the putative thaumatin's receptor. Less
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