A study of the relationships of interactions between Asp-201, Na⁺or K⁺, and galactosyl C6 hydroxyl and their effects on binding and reactivity of β-galactosidase

The interactions between Na + (and K + ) and Asp-201 of β-galactosidase were studied. Analysis of the changes in K m and V max showed that the K d for Na + of wild type β-galactosidase (0.36 ± 0.09 mM) was about 10× 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 K m as functions of Na + and K + for oNPG and pNPG and decreases of the K i of both shallow and deep mode inhibitors were similar, whereas the K m and K i 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 V max . The V max 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 k 3 (degalactosylation) to increase. Na + also caused the k 2 (galactosylation) with oNPG, but not with pNPG, to increase. In contrast, K + caused the k 2 values with both oNPG and pNPG to increase. Na + and K + mainly altered the entropies of activation of k 2 and k 3 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 K m and K i values to increase significantly. In addition, the K d 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 K m and K i values of D201N-β-galactosidase and the V max values changed minimally and in a linear rather than hyperbolic way. D201F-β-galactosidase, with a very bulky hydrophobic side chain in place of Asp, essentially obliterated all binding and catalysis.Key words: β-galactosidase, sodium, potassium, binding, aspartic acid.