Martinez-Bilbao M, Huber R E
Faculty of Science, University of Calgary, Canada.
Biochem Cell Biol. 1996;74(2):295-8. doi: 10.1139/o96-032.
The activation of beta-galactosidase (E. coli) by Mg(2+) at pH values below 7.6 was studied. If the Mg(2+) concentration was high enough, the k(cat) values at pH values down to 5.0 remained at the same high level as at pH 7 and 7.6 (600-620 s-(1) with o-nitrophenyl-beta-D-galactopyranoside as the substrate). Very high concentrations of Mg(2+) (greater than 100 mM at pH 5) were, however, needed to saturate the Mg(2+) site at lower pH values. The Km values at low levels of Mg(2+) were high at every pH but they decreased and approached the same low value at every pH (about 0.13 mM) as the [Mg(2+)] was increased. These data indicate that it is difficult to bind Mg(2+) at lower pH values, but the k(cat) and K(m) values of the enzyme, and therefore the rates of galactosylation (k(2)), degalactosylation (k(3)), and binding (K(s)), do not change substantially as a function of pH provided that a Mg(2+) is bound to the enzyme. The data also showed that Mg(2+) and protons compete for the same site. Analysis by plotting log Mg(2+) vs. pH showed that the binding of Mg(2+) to the free enzyme involves two groups with pK(a) values in the vicinity of 7 and one group with a pK(a) value near 5.5. (The values referred to as Mg(2+) are the Mg(2+) concentrations that resulted in k(cat) values midway between basal and maximum.) The "apparent" pK(a) values of the groups when a Mg(2+) was bound (at saturating [Mg(2+)]) all appeared to be below 5.0.
研究了在pH值低于7.6时镁离子（Mg(2+)）对β-半乳糖苷酶（大肠杆菌）的激活作用。如果Mg(2+)浓度足够高，在pH值低至5.0时的k(cat)值与在pH 7和7.6时保持在相同的高水平（以邻硝基苯基-β-D-吡喃半乳糖苷为底物时为600 - 620 s-(1)）。然而，在较低pH值下需要非常高浓度的Mg(2+)（在pH 5时大于100 mM）才能使Mg(2+)位点饱和。在低水平Mg(2+)时，每个pH值下的Km值都很高，但随着[Mg(2+)]的增加，它们会降低并在每个pH值下接近相同的低值（约0.13 mM）。这些数据表明，在较低pH值下难以结合Mg(2+)，但只要有Mg(2+)结合到酶上，酶的k(cat)和K(m)值，以及因此半乳糖基化速率（k(2)）、去半乳糖基化速率（k(3)）和结合速率（K(s)），不会随pH值有显著变化。数据还表明Mg(2+)和质子竞争相同的位点。通过绘制log Mg(2+)对pH的图进行分析表明，Mg(2+)与游离酶的结合涉及两个pK(a)值在7附近的基团和一个pK(a)值接近5.5的基团。（称为Mg(2+)的值是导致k(cat)值处于基础值和最大值中间的Mg(2+)浓度。）当结合Mg(2+)（在饱和[Mg(2+)]时）时，这些基团的“表观”pK(a)值似乎都低于5.0。
