Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase
Structural and kinetic characterization of Escherichia coli TadA, the wobble-specific tRNA deaminase
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DOI：
10.1021/bi0522394
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发表时间：
2006-05-23
期刊：
影响因子：
2.9
通讯作者：
Almo, Steven C.
中科院分区：
文献类型：
--
作者：
Kim, Jungwook;Malashkevich, Vladimir;Almo, Steven C.
必需的tRNA特异性腺苷脱氨酶催化tRNA摆动位置的腺苷脱氨为肌苷。这种修改允许单个tRNA物种识别在最后(3‘-大多数)位置包含A、C或U的多个同义密码子，并确保所有正义密码子都被适当地解码。我们首次报道了摇摆特异性脱氨酶的结构和动力学相结合的特征。大肠杆菌酶的结构清楚地定义了二聚体的界面和催化必需的锌离子的配位。该结构还识别了亲核水，并突出了催化锌附近的残基，这些残基可能参与识别和催化聚合RNA底物。最小的19个核苷酸的RNA茎底物允许该酶的第一个稳态动力学特征(k(Cat)=13+/-1min(-1)，K-M=0.83+/-0.22AM)。建立了一种在高浓度多核苷酸底物(>；10muM)下跟踪反应的连续偶联分析方法。这项工作开始定义负责催化和底物识别的化学和结构决定因素，并为这种关键酶的详细机制分析奠定基础。
The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k(cat) = 13 +/- 1 min(-1) and K-M = 0.83 +/- 0.22 AM). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (> 10 mu M). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.