KINETICS OF DISULFIDE BOND REDUCTION IN ALPHA-LACTALBUMIN BY DITHIOTHREITOL AND MOLECULAR-BASIS OF SUPERREACTIVITY OF THE CYS6-CYS120 DISULFIDE BOND
KINETICS OF DISULFIDE BOND REDUCTION IN ALPHA-LACTALBUMIN BY DITHIOTHREITOL AND MOLECULAR-BASIS OF SUPERREACTIVITY OF THE CYS6-CYS120 DISULFIDE BOND
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DOI：
10.1021/bi00488a007
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发表时间：
1990-09-11
期刊：
影响因子：
2.9
通讯作者：
SUGAI, S
中科院分区：
文献类型：
--
作者：
KUWAJIMA, K;IKEGUCHI, M;SUGAI, S
研究了二硫苏糖醇还原α-乳清蛋白中二硫代谢物的动力学，测量了在310 nm处的吸收光谱和270 nm处(pH 8.5或7.0，25℃)的CD椭圆度随时间的变化。当二硫键完整的蛋白质折叠时，动力学是两相的。半胱氨酸-6和-120之间的二硫键在快相被还原，其他三个二硫键在慢相被还原。两相的表观速率常数均与二硫苏糖醇的浓度成正比，表明两相均为生物分子反应。然而，决定反应速率的详细分子机制在两个阶段之间明显不同。慢相显示出随着变性剂尿素浓度的增加反应速度呈S形增加，并且也被蛋白质中结合的钙离子去除时天然状态的不稳定所加速。在天然结构中，二硫键明显受到保护，不受还原剂的影响。然而，快相反应速率随尿素浓度的增加而降低，二硫键在自然条件下表现出极高的反应活性。它在完全可及状态下的活性是正常二硫化物的140倍，并且由快相产生的三-二硫代α-乳清蛋白在强烈的自然条件下具有天然结构。由于离子强度不影响这种二硫键的超活性，静电对反应活性的贡献必须可以忽略不计。基于狒狒α-乳清蛋白的精细X射线坐标的二硫键几何构型的检查[Acharya等人。(1989)J.Mol.比奥尔。208，99-127]，并与其他五种蛋白质的几何结构比较清楚地表明，超反应活性源于天然结构折叠施加在二硫键上的几何应变。讨论了二硫键应变能与蛋白质稳定性和二硫键反应活性的关系。
Kinetics of disulfide reduction in .alpha.-lactalbumin by dithiothreitol are investigated by measuring time-dependent changes in absorption at 310 nm and in CD ellipticity at 270 nm (pH 8.5 or 7.0, and 25.degree.C). When the disulfide-intact protein is folded, the kinetics are biphasic. The disulfide bond between the half-cystines-6 and -120 is reduced in the fast phase, and the other three disulfide bonds are reduced in the slow phase. The apparent rate constants of the two phases are both proportional to the concentration of dithiothreitol, indicating that both phases are expressed by biomolecular reactions. However, detailed molecular mechanisms that determine the reaction rates are markedly different between the two phases. The slow phase shows a sigmoidal increase in the reaction rate with increasing concentration of a denaturant, urea, and is also accelerated by destabilization of the native state on removal of the bound Ca2+ ion in the protein. The disulfide bonds are apparently protected against the reducing agent in the native structure. The fast phase reaction rate is, however, decreased with an increase in the concentration of urea, and the disulfide bond shows extraordinary superreactivity in native conditions. It is 140 times more reactive than normal disulfides in the fully accessible state, and three-disulfide .alpha.-lactalbumin produced by the fast phase assumes nativelike structure under a strongly native condition. As ionic strength does not affect the superreactivity of this disulfide bond, electrostatic contributions to the reactivity must be negligible. Inspection of the disulfide bond geometry based on the refined X-ray coordinates of baboon .alpha.-lactalbumin [Acharya et al. (1989) J. Mol. Biol. 208, 99-127] and comparison of the geometry with those in five other proteins clearly demonstrate that the superreactivity arises from the geometric strain imposed on this disulfide bond by the native structure folding. Relationships of the disulfide strain energy to the protein stability and the disulfide reactivity are discussed.