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

Abstract

The interactions between Na+(and K+) and Asp-201 of β-galactosidase were studied. Analysis of the changes in Kmand Vmaxshowed that the Kdfor 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 Kmas functions of Na+and K+for oNPG and pNPG and decreases of the Kiof both shallow and deep mode inhibitors were similar, whereas the Kmand Kiof 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 Vmaxof 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 k2values with both oNPG and pNPG to increase. Na+and K+mainly altered the entropies of activation of k2and k3with 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 Kmand Kivalues to increase significantly. In addition, the Kdvalues 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 Kmand Kivalues of D201N-β-galactosidase and the Vmaxvalues 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.