甘味タンパク質の甘味活性発現機構

Thaumatin是一种不含半胱氨酸残基和8个分子内二硫键的甜蛋白，在70℃以上的pH 7.0加热时聚集，其甜味随之消失。在硫醇还原剂和SDS存在的情况下，聚集体可以通过加热实现溶解。这种分子聚集取决于加热过程中的蛋白质浓度，并被n -乙基马来酰亚胺或碘乙酰胺抑制，表明热变性分子之间发生了硫醇催化的二硫交换反应。对聚集体的氨基酸分析表明，半胱氨酸和赖氨酸残基被还原，并形成了半胱氨酸残基和赖氨酸残基。这些残基的还原和形成在化学计量上满足胱氨酸残基的b消除。半胱氨酸催化了二硫交换反应；即，通过二硫键的b消去形成游离巯基残基。在pH 7.0下加热后，分子间可能会形成更多的ide键，导致分子聚集。Thaumatin I是一种甜味蛋白质，从一种原产于热带西非的植物的果皮中分离出来。然而，尽管它的甜味很强，但其甜味的结构基础仍然未知。通过化学修饰研究，确定了具有重要功能的赖氨酸残基。用吡哆醛5′-磷酸（Pyridoxal 5′-phosphate， PLP）选择性修饰thumatin I上的赖氨酸残基，PLP与thumatin I在pH 7.0下孵育，用硼氢化钠还原席夫碱。纯化了5个修饰的thaumatin I，每个thaumatin I分子都含有1个PLP，并鉴定了其修饰的赖氨酸残基。对Lys78、lys97、Lys106、Lys137和Lys187进行了修饰，从圆二色光谱的测量结果可以看出，这些修饰均不影响thumatin I的构象。改良后的thaumatin I甜度降低，损失67-85%。修饰后的thaumatin I用磷酸酶处理去除附着的PLP的磷酸基团。从Lys78-、Lys97-、Lys137-和lys187 -修饰的thaumatin I中去除后，恢复了与天然thaumatin I相当的强烈甜味，而从lys106 -修饰的thaumatin I中去除后，则无法恢复甜味。这些结果表明，Lys78、Lys97、Lys106、Lys137和Lys187这5个赖氨酸残基具有重要的功能。我们的研究结果还表明，尽管Lys106的作用尚不清楚，但Lys78、Lys97、Lys137和Lys187的正电荷参与了与假定的突变蛋白受体的静电相互作用。少

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 … More 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