Sun A Q, Yüksel K U, Gracy R W
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107, USA.
Arch Biochem Biophys. 1995 Oct 1;322(2):361-8. doi: 10.1006/abbi.1995.1476.
Mammalian triosephosphate isomerase spontaneously deamidates at Asn71 and Asn15 located at the subunit interface of the isologous dimer. These deamidations have been proposed to constitute the terminal marking event in the degradation of the enzyme. A series of physical and chemical studies detailed here reveals that the overall structure of the enzyme is substantially altered by these deamidations. The far-uv CD spectra show a 30% lower secondary structure with a blue shifted ellipticity minimum and increased fluorescence (10-22%) with a red-shifted emission maximum (8.7-15.6 nm) indicates exposure of tryptophans to a more polar environment. Increased binding of the fluorescent hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid to the deamidated enzyme corroborates these spectral observations and also suggests that the hydrophobic residues at the subunit interface are exposed as a result of the deamidation. Decreased subunit cross-linking (80 vs 20%) of the deamidated enzyme by the bifunctional reagent ethylene glycolbis (succinimidylsuccinate) also indicates a loosening of the two subunits at the interface. These structural changes are accompanied by a decreased thermal stability (3.1 degrees C lower Tm) and an increased susceptibility to dissociation in urea. The terminal marking also results in the generation of new proteolytic sites and increases the susceptibility to proteolysis. Hybrid dimers from rabbit and yeast (lacking Asn71) showed that deamidation of the rabbit Asn71-yeast Asn15 pair does not accelerate deamidation of the remaining rabbit Asn15 site, indicating that deamidation of Asn71 is a prerequisite for deamidation of Asn15. These studies are consistent with the proposal that the specific deamidations at the subunit interface cause significant structural changes which lead to degradation of the protein.
哺乳动物磷酸丙糖异构酶在位于同源二聚体亚基界面的Asn71和Asn15处自发脱酰胺。这些脱酰胺作用被认为是该酶降解过程中的终末标记事件。此处详细介绍的一系列物理和化学研究表明，这些脱酰胺作用会使酶的整体结构发生显著改变。远紫外圆二色光谱显示二级结构降低了30%，椭圆率最小值发生蓝移，荧光增强（10 - 22%），发射最大值红移（8.7 - 15.6 nm），这表明色氨酸暴露于更具极性的环境中。荧光疏水探针1,1'-双（4-苯胺基）-萘-5,5'-二磺酸与脱酰胺酶的结合增加，证实了这些光谱观察结果，也表明亚基界面处的疏水残基因脱酰胺作用而暴露。双功能试剂乙二醇双（琥珀酰亚胺琥珀酸酯）对脱酰胺酶的亚基交联减少（80%对20%），这也表明界面处的两个亚基发生了松散。这些结构变化伴随着热稳定性降低（熔解温度低3.1℃）以及在尿素中解离敏感性增加。终末标记还导致产生新的蛋白水解位点并增加对蛋白水解的敏感性。来自兔子和酵母（缺乏Asn71）的杂合二聚体表明，兔子Asn71 - 酵母Asn15对的脱酰胺作用不会加速其余兔子Asn15位点的脱酰胺作用，这表明Asn71的脱酰胺作用是Asn15脱酰胺作用的先决条件。这些研究与以下观点一致，即亚基界面处的特定脱酰胺作用会导致显著的结构变化，从而导致蛋白质降解。
