Amino acid substitutions at the subunit interface of dimeric escherichia coli alkaline phosphatase cause reduced structural stability
Amino acid substitutions at the subunit interface of dimeric escherichia coli alkaline phosphatase cause reduced structural stability
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二聚体大肠杆菌碱性磷酸酶亚基界面的氨基酸取代导致结构稳定性降低
DOI：
10.1110/ps.8.5.1152
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发表时间：
1999
期刊：
影响因子：
8
通讯作者：
E. Kantrowitz
中科院分区：
文献类型：
--
作者：
Donna Carolan Martin;S. Pastra;E. Kantrowitz
研究了二聚体界面氨基酸取代对单体间相互作用强度的影响以及对来自大肠杆菌的二聚体酶碱性磷酸酶催化功能的影响。用动力学分析、超速离心法和横向尿素梯度凝胶电泳法对二聚体界面氨基酸取代的R10A、R10K、R24A、R24K、T59A和R10A/R24A酶进行了鉴定。野生型和变异型碱性磷酸酶的动力学数据显示出相似的催化行为，其kcat值在51.3%到69.5%之间，Km值在14.8%到26.3kgM之间。超速离心法表明野生型酶比所有界面修饰酶更稳定。该野生型酶在pH 4.0及以上范围内为二聚体，在pH 3.5及以下时可分解。然而，所有界面修饰的酶在pH 4.0时明显是单体，在pH 5.0开始组装，直到pH 6.0才完全组装成二聚体。横向尿素梯度凝胶电泳法的结果显示，这些修饰的酶对变性剂更加敏感，在1.0~1.5M的尿素浓度范围内开始展开，在2.5M以上，野生型酶仍以折叠的高迁移率形式存在。碱性磷酸酶H370A在活性部位修饰，而不是在二聚体界面修饰，在超速离心法和电泳法研究中都与野生型酶相似。结果表明，界面上单一氨基酸的取代不仅牺牲了组装的二聚体的完整性，而且牺牲了单体折叠的稳定性，即使在最适pH下酶的活性不受影响，似乎也不依赖于界面的稳定性。
The consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild‐type and altered alkaline phosphatases show comparable catalytic behavior with kcat values between 51.3 and 69.5 s–1 and Km values between 14.8 and 26.3 μM. The ultracentrifugation profiles indicate that the wild‐type enzyme is more stable than all the interface‐modified enzymes. The wild‐type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface‐modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild‐type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild‐type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.
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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。
影响因子：
2.9
作者：
Xu,X;Kantrowitz,ER
通讯作者：
Kantrowitz,ER
大肠杆菌碱性磷酸酶的精制结构，分辨率为 2.8 A。
影响因子：
5.6
作者：
Sowadski,JM;Handschumacher,MD;Murthy,HM;Foster,BA;Wyckoff,HW
通讯作者：
Wyckoff,HW
影响因子：
14.9
作者：
HIGUCHI, R;KRUMMEL, B;SAIKI, RK
通讯作者：
SAIKI, RK
DOI：
10.1073/pnas.78.6.3473
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
BRADSHAW, RA;CANCEDDA, F;WALSH, KA
通讯作者：
WALSH, KA