Cathala G, Brunel C
J Biol Chem. 1975 Aug 10;250(15):6046-53.
Kidney alkaline phosphatase is an enzyme which requires two types of metals for maximal activity: zinc, which is essential, and magnesium, which is stimulatory. The main features of the Mg2+ stimulation have been analyzed. The stimulation is pH-dependent and is observed mainly between pH 7.5 and 10.5. Mg2+ binding to native alkaline phosphatase is characterized by a dissociation constant of 50 muM at pH 8.5,25 degrees. Binding of Zn2+ is an athermic process. Both the rate constants of association, ka, and of dissociation, kd, have low values. Typical values are 7 M(-1) at pH 8.0, 25 degrees, for ka and 4.10(-4) S(-1) at pH 8.0, 25 degrees, for kd. The on and off processes have high activation energies of 29 kcal mol (-1). Mg2+ can be replaced at its specific site by Mn2+, Co2+, Ni2+, and Zn2+. Zinc binding to the Mg2+ site inhibits the native alkaline phosphatase. Mn2+, Co2+, and Ni2+ also bind to the Mg2+ site with a stimulatory effect which is nearly identic-al with that of Mg2+, Mn2+ is the stimulatory cation which binds most tightly to the Mg2+ site; the dissociation constant of the Mn2+ kidney phosphatase complex is 2 muM at pH 8.5. The stoichiometry of Mn2+ binding has been found to be 1 eq of Mn2+ per mol of dimeric kidney phosphatase. The native enzyme displays absolute half-site reactivity for Mn2+ binding. Mg2+ binding site and the substrate binding sites are distinct sites. The Mg2+ stimulation corresponds to an allosteric effect. Mg2+ binding to its specific sites does not affect substrate recognition, it selectively affects Vmax values. Quenching of the phosphoenzyme formed under steady state conditions with [32P]AMP as a substrate as well as stopped flow analysis of the catalyzed hydrolysis of 2,4-dinitrophenyl phosphate or p-nitrophenyl phosphate have shown that the two active sites of the native and of the Mg2+-stimulated enzyme are not equivalent. Stopped flow analysis indicated that one of the two active sites was phosphorylated very rapidly whereas the other one was phosphorylated much more slowly at pH 4.2. Half of the sites were shown to be reactive at pH 8.0. Quenching experiments have shown that only one of the two sites is phosphorylated at any instant; this result was confirmed by the stopped flow observation of a burst of only 1 mol of nitrophenol per mol of dimeric phosphatase in the pre-steady state hydrolysis of p-nitrophenyl phosphate. The half-of-the-sites reactivity observed for the native and for the Mg2+-stimulated enzyme indicates that the same type of complex, the monophosphorylated complex, accumulates under steady state conditions with both types of enzymes. Mg2+ binding to the native enzyme at pH 8.0 increases considerably the dephosphorylation rate of this monophosphorylated intermediate. A possible mechanism of Mg2+ stimulation is discussed.
必不可少的锌和起刺激作用的镁。已对Mg2+刺激的主要特征进行了分析。这种刺激是pH依赖性的，主要在pH 7.5至10.5之间观察到。在pH 8.5、25℃时，Mg2+与天然碱性磷酸酶结合的解离常数为50μM。Zn2+的结合是一个无热过程。缔合速率常数ka和解离速率常数kd的值都很低。在pH 8.0、25℃时，ka的典型值为7 M-1，kd的典型值为4×10-4 S-1。结合和解离过程具有29 kcal·mol-1的高活化能。Mg2+在其特定位点可被Mn2+、Co2+、Ni2+和Zn2+取代。锌与Mg2+位点的结合会抑制天然碱性磷酸酶。Mn2+、Co2+和Ni2+也与Mg2+位点结合，具有与Mg2+几乎相同的刺激作用；Mn2+是与Mg2+位点结合最紧密的刺激阳离子；在pH 8.5时，Mn2+与肾磷酸酶复合物的解离常数为2μM。已发现Mn2+结合的化学计量比为每摩尔二聚体肾磷酸酶1当量的Mn2+。天然酶对Mn2+结合表现出绝对的半位点反应性。Mg2+结合位点和底物结合位点是不同的位点。Mg2+刺激对应于一种别构效应。Mg2+与其特定位点的结合不影响底物识别，它选择性地影响Vmax值。用[32P]AMP作为底物对稳态条件下形成的磷酸酶进行淬灭，以及对2,4-二硝基苯磷酸酯或对硝基苯磷酸酯催化水解的停流分析表明，天然酶和Mg2+刺激酶的两个活性位点并不等效。停流分析表明，在pH 4.2时，两个活性位点中的一个非常迅速地被磷酸化，而另一个则被磷酸化得慢得多。在pH 8.0时，一半的位点显示有反应性。淬灭实验表明，在任何时刻，两个位点中只有一个被磷酸化；在对硝基苯磷酸酯的预稳态水解中，每摩尔二聚体磷酸酶仅释放1摩尔硝基苯酚的停流观察结果证实了这一结果。天然酶和Mg2+刺激酶的半位点反应性表明，在稳态条件下，两种类型的酶都会积累相同类型的复合物，即单磷酸化复合物。在pH 8.0时，Mg2+与天然酶的结合会显著提高这种单磷酸化中间体的去磷酸化速率。讨论了Mg2+刺激的一种可能机制。
