Xu Julia, McRae Mary A A, Harron Scott, Rob Beatrice, Huber Reuben E
Division of Biochemistry, University of Calfgary, Alberta, Canada.
Biochem Cell Biol. 2004 Apr;82(2):275-84. doi: 10.1139/o04-004.
The interactions between Na+ (and K+) and Asp-201 of beta-galactosidase were studied. Analysis of the changes in Km and Vmax showed that the Kd for Na+ of wild type beta-galactosidase (0.36 +/- 0.09 mM) was about 10x lower than for K+ (3.9 +/- 0.6 mM). The difference is probably because of the size and other physical properties of the ions and the binding pocket. Decreases of Km as functions of Na+ and K+ for oNPG and pNPG and decreases of the Ki of both shallow and deep mode inhibitors were similar, whereas the Km and Ki of substrates and inhibitors without C6 hydroxyls remained constant. Thus, Na+ and K+ are important for binding galactosyl moieties via the C6 hydroxyl throughout catalysis. Na+ and K+ had lesser effects on the Vmax. The Vmax of pNPF and pNPA (substrates that lack a C6 hydroxyl) did not change upon addition of Na+ or K+, showing that the catalytic effects are also mediated via the C6 hydroxyl. Arrhenius plots indicated that Na+, but not K+, caused k3 (degalactosylation) to increase. Na+ also caused the k2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K+ caused the k2 values with both oNPG and pNPG to increase. Na+ and K+ mainly altered the entropies of activation of k2 and k3 with only small effects on the enthalpies of activation. This strongly suggests that only the positioning of the substrate, transition states, and covalent intermediate are altered by Na+ and K+. Further evidence that positioning is important was that substitution of Asp-201 with a Glu caused the Km and Ki values to increase significantly. In addition, the Kd values for Na+ or K+ were 5 to 8 fold higher. The negative charge of Asp-201 was shown to be vital for Na+ and K+ binding. Large amounts of Na+ or K+ had no effect on the very large Km and Ki values of D201N-beta-galactosidase and the Vmax values changed minimally and in a linear rather than hyperbolic way. D201F-beta-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.
研究了β-半乳糖苷酶中Na⁺（和K⁺）与天冬氨酸-201之间的相互作用。对Km和Vmax变化的分析表明，野生型β-半乳糖苷酶对Na⁺的解离常数（Kd）（0.36±0.09 mM）比对K⁺的解离常数（3.9±0.6 mM）低约10倍。这种差异可能是由于离子的大小和其他物理性质以及结合口袋所致。对于邻硝基苯-β-D-半乳糖苷（oNPG）和对硝基苯-β-D-半乳糖苷（pNPG），Km随Na⁺和K⁺的降低以及浅模式和深模式抑制剂的抑制常数（Ki）的降低相似，而没有C6羟基的底物和抑制剂的Km和Ki保持不变。因此，在整个催化过程中，Na⁺和K⁺对于通过C6羟基结合半乳糖基部分很重要。Na⁺和K⁺对Vmax的影响较小。添加Na⁺或K⁺后，对硝基苯-β-D-半乳糖苷（pNPF）和对硝基苯-α-D-半乳糖苷（pNPA）（缺乏C6羟基的底物）的Vmax没有变化，表明催化作用也是通过C6羟基介导的。阿累尼乌斯曲线表明，Na⁺而非K⁺导致k3（脱半乳糖基化）增加。Na⁺还导致oNPG的k2（半乳糖基化）增加，但pNPG的k2没有增加。相反，K⁺导致oNPG和pNPG的k2值均增加。Na⁺和K⁺主要改变了k2和k3的活化熵，对活化焓的影响很小。这有力地表明，只有底物、过渡态和共价中间体的定位被Na⁺和K⁺改变。定位很重要的进一步证据是，用谷氨酸替代天冬氨酸-201导致Km和Ki值显著增加。此外，对Na⁺或K⁺的Kd值高5至8倍。天冬氨酸-201的负电荷对于Na⁺和K⁺的结合至关重要。大量的Na⁺或K⁺对D201N-β-半乳糖苷酶非常大的Km和Ki值没有影响，Vmax值变化很小且呈线性而非双曲线方式。D201F-β-半乳糖苷酶用非常庞大的疏水侧链取代天冬氨酸，基本上消除了所有结合和催化作用。
