Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis.
Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis.
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通过定点诱变探讨组氨酸 372 在锌结合中的作用以及大肠杆菌碱性磷酸酶的催化机制。
DOI：
10.1021/bi00174a039
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发表时间：
1994
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;Qin,XQ;Kantrowitz,ER
摘要：在2.0-A分辨率的大肠杆菌碱性磷酸酶的X射线结构中，发现His-372距离锌仅3.8 A，并与Asp-327形成氢键相互作用，Asp-327是锌在Ml位点的双齿配体。然而，His-372不直接与M1位点的锌原子相互作用。为了研究His-372侧链在锌结合中的作用以及大肠杆菌碱性磷酸酶的催化机制，采用定点突变的方法将His-372转化为丙氨酸。His-372-* Ala酶具有与野生型酶相似的锌结合亲和力的事实表明His-372不参与Ml位点处的锌结合。然而，与野生型酶相比，突变酶的动力学行为改变，表明His-372的咪唑环在酶的催化机制中起间接作用。His-372-* Ala酶在pH 8.0下的水解活性比野生型酶低10倍。在pH 8.0的磷酸盐受体的存在下，突变酶的活性约为野生型酶的80%。因此，His-372-Ala突变选择性地增强了酶的转磷酸化活性。His-372-* Ala酶在0.1 M MOPS缓冲液和1.0 M Tris缓冲液（pH 8.0）中的Km也分别比野生型酶降低4倍和30倍。与野生型酶相比，还观察到His-372-* Ala酶在pH 8.0下的速率决定步骤的变化。在His-372-* Ala酶的预稳态动力学中存在瞬时爆发表明共价磷酸丝氨酸键的断裂是反应中的限速步骤。在pH 8.0下，[32 P] P1没有掺入His-372-* Ala酶中，表明在从酶-磷酸盐共价复合物到酶-磷酸盐非共价复合物的过渡期间存在稳定的中间体。综上所述，这些动力学结果表明，在His-372-* Ala酶中，与Ml位点处的锌原子配位的羟基是比野生型酶中更弱的亲核试剂。因此，His-372和Asp-327的侧链之间的相互作用对于稳定锌羟基可能是重要的，锌羟基是负责磷酸丝氨酸中间体的分解的亲核基团。1.3. 1)催化磷酸单酯的非特异性水解（Reid & Wilson，1971）。该酶还可以催化转磷酸化反应，其中磷酸从磷酸单酯转移到醇（Dayan & Wilson，1964; Wilson et al.，1964年）。在大肠在大肠杆菌中，碱性磷酸酶是二聚金属酶，每个单体含有两个紧密结合的锌原子和一个镁原子。酶促反应的机制已经被彻底研究，并且已经分离出具有Ser-102磷酸化的稳定的磷酸酶中间体（Schwartz & Lipmann，1961; Engstrom，1962; Schwartz等人，1963年）。许多动力学和物理化学研究表明，在酸性pH下，共价磷酸丝氨酸中间体（EP 1）水解形成非共价酶-磷酸盐复合物（E-Pi）是限速步骤，而在碱性pH下，E-Pi复合物的解离是限速步骤（船体等人，1976; Reid & Wilson，1971; Gettins &科尔曼，1983 a; Gettins等人，1985; Bloch & Gorby，1980）。X射线结构分析表明，E.大肠杆菌碱性磷酸酶已被测定并精确到2.0（Kim和维科夫，1989，
Revised Manuscript Received November 29, 1993® abstract: In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the Ml site. However, His-372 does not directly interact with the zinc atom at the M1 site. In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-* Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the Ml site. However, the alteredkinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of theenzyme. The hydrolysis activity of the His-372-* Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme. In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme. Therefore, the His-372—Ala mutation selectively enhances the transphosphorylation activity of the enzyme. The His-372-* Ala enzyme also exhibits 4-and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively. A change in the rate-determining step at pH 8.0 is also observed for the His-372-* Ala enzyme compared with the wild-type enzyme. The presence of a transient burst in the pre-steady-state kinetics of the His-372-* Ala enzyme indicates that breaking of the covalent phosphoserine bond is the rate-limiting step in the reaction. The lack of incorporation of [32P] P; into the His-372-* Ala enzyme at pH 8.0 suggests the presence of a stable intermediate during the transition from the enzyme-phosphate covalent complex to the enzyme-phosphate noncovalent complex. Taken together, these kinetic results suggest that the hydroxyl group coordinatedto the zinc atom at the Ml site is a weaker nucleophile in the His-372—* Ala enzyme than in the wild-type enzyme. Therefore, the interaction between the side chain of His-372 and Asp-327 may be important for stabilizing the zinc hydroxyl which is the nucleophilic group that is responsible for the breakdown of the phosphoserine intermediate.Alkaline phosphatase isolated from the periplasmic space of Escherichia coli (EC3. 1.3. 1) catalyzes the nonspecific hydrolysis of phosphomonoesters (Reid & Wilson, 1971). The enzyme can also catalyze a transphosphorylation reaction with the transfer of the phosphate from the phosphomonoester to an alcohol (Dayan & Wilson, 1964; Wilson et al., 1964). In E. coli, alkaline phosphatase is a dimeric metalloenzyme, containing two tightly bound zinc atoms and one magnesium atom per monomer. The mechanism of the enzymatic reaction has been thoroughly studied, and a stable phosphoenzyme intermediate with Ser-102 phosphorylated has been isolated (Schwartz & Lipmann, 1961; Engstrom, 1962; Schwartz et al., 1963). Many kinetic and physicochemical studies suggest that, at acidic pH, the hydrolysis of the covalent phosphoserine intermediate (EP,) to form a noncovalent enzyme-phosphate complex (E-Pi) is the rate-limiting step while at alkaline pH the dissociation of the E-Pj complex is the rate-limiting step (Hull et al., 1976; Reid & Wilson, 1971; Gettins & Coleman, 1983a; Gettins et al., 1985; Bloch & Gorby, 1980). The X-ray structure of E. coli alkaline phosphatase has been determined and refined to 2.0 A (Kim & Wyckoff, 1989,
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DOI：
--
发表时间：
1963
影响因子：
11.1
作者：
J. H. Schwartz;A. M. Crestfield;F. Lipmann
通讯作者：
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DOI：
10.2210/pdb1anh/pdb
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Murphy,JE;Xu,X;Kantrowitz,ER
通讯作者：
Kantrowitz,ER
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影响因子：
2.9
作者：
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通讯作者：
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影响因子：
2.9
作者：
Xu,X;Kantrowitz,ER
通讯作者：
Kantrowitz,ER
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DOI：
--
发表时间：
1992
期刊：
影响因子：
--
作者：
S. Mangani;P. Carloni;M. Viezzoli;J. Coleman
通讯作者：
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