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首页> 外文期刊>Biochemistry >Zinc Ion Effects on Individual Ssp DnaE Intein Splicing Steps: Regulating Pathway Progression.
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Zinc Ion Effects on Individual Ssp DnaE Intein Splicing Steps: Regulating Pathway Progression.

机译：锌离子对单个Ssp DnaE内含子剪接步骤的影响：调节途径进程。

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Use of the naturally split, self-splicing Synechocystis sp. PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains. Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics. Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions. Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry. Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2). Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2). These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the(+)1 cysteine residue. N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack. Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined. Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step. Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA. By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.
机译：使用自然分裂的，自分裂的Synechocystis sp。 PCC6803 DnaE内含子允许分别纯化N和C末端内含子结构域。否则，可以在体外控制自发的内含蛋白介导的反应，从而可以详细研究内含蛋白的动力学。 Ssp DnaE内含子与ZnCl（2）的孵育抑制反式剪接，水解介导的N末端反式裂解和C末端反式裂解反应。在等摩尔浓度的ZnCl（2）和内含肽域，达到1：1的金属离子：内含肽结合化学计量，实现了最大的抑制的剪接反应。 （+）1半胱氨酸残基突变为缬氨酸（C（+）1V）减轻了ZnCl（2）的抑制作用。在没有ZnCl（2）的情况下缬氨酸取代会阻止反式剪接，并以类似于存在ZnCl（2）的天然（+）1半胱氨酸的方式降低C末端裂解动力学。这些数据与通过（+）1半胱氨酸残基的螯合对Ssp DnaE内含子的Zn（2+）介导的抑制作用一致。 N端反式裂解可通过自发水解和亲核（例如DTT）攻击发生。使用氨基酸取代（C（+）1V）和Zn（2+）介导的抑制作用的N末端裂解率的比较检查可以确定水解对整个N末端裂解动力学的最大贡献。通过PAGE检测到由相关的内含蛋白结构域组成的稳定中间体，并提供了快速C末端裂解步骤的证据。通过快速逆转EDTA对Zn（2+）介导的抑制作用，可以实现C末端反应的急性控制。通过抑制剪接途径和Zn（2+）的自发水解，反应物可以从反式剪切途径转移到反式剪切途径，其中DTT和EDTA分别调节N和C末端的裂解。

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