A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity.
A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity.
复制标题
大肠杆菌碱性磷酸酶催化位点中水介导的盐连接可能会影响活性。
DOI：
10.1021/bi00245a018
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Kantrowitz,ER
中科院分区：
文献类型：
--
作者：
Xu,X;Kantrowitz,ER
波士顿学院化学系，切斯特纳特山，马萨诸塞州02167接收于1991年2月19日;修订于1991年5月9日摘要：大肠杆菌碱性磷酸酶以相似的速率催化多种磷酸单酯的水解，反应通过磷酸酶中间体进行。活性位点区域在E.大肠杆菌和大肠杆菌碱性磷酸酶。E.大肠杆菌酶的结果表明，在所有哺乳动物碱性磷酸酶中被组氨酸取代的Lys-328通过水分子桥连到磷酸上。该水分子也与Asp-327（两个锌原子之一的双齿配体）氢键结合。在这里，我们报告使用位点特异性诱变转换赖氨酸-328组氨酸和丙氨酸。高于pH 7.0的稳态动力学研究表明，这两种突变酶的pH值与活性曲线相比，野生型酶的曲线发生了变化。在pH 10.3，在Tris存在下，Lys-328-*Ala酶的活性比野生型酶高约14倍。在相同的pH值下，在不存在Tris的情况下，Lys-328-Ala酶的活性仍然比野生型酶高6倍。这两种突变体酶具有较低的磷酸盐亲和力比野生型酶在所有pH值的调查。在pH 5.5下的预稳态动力学揭示了Lys-328 Ala酶的行为与无磷酸盐的野生型酶非常相似。然而，在pH8.0时，与野生型酶不表现出瞬态相反，突变型酶在稳态前表现出一个小的瞬态，表明该突变型酶在此pH下的限速步骤可能发生变化。突变酶的性质可以根据磷酸盐亲和力的降低来合理化。这些数据还表明锌配位羟基的pKa可能发生变化。
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02167 Received February 19, 1991; Revised Manuscript Received May 9, 1991 abstract: Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalianalkaline phosphatases. The three-di-mensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine inall mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profilescompared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328-*Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH inthe absence of Tris the Lys-328—Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328 Ala enzyme behaves very similar to the phosphate-free wild-type enzyme. However, at pH 8.0, as opposed to the wild-type enzyme that does not exhibit a transient phase, the mutant enzyme shows a small transient phase in thepre steady state, suggesting a possible change in the rate-limiting step for this mutant enzyme at thispH. The properties of the mutant enzymes can be rationalized on the basis of a reduction in phosphate affinity. These data also suggest a possible alteration of the pKa of the zinc-coordinated hydroxyl group.