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首页> 外文期刊>Chemistry: A European journal >Binding and inhibition of copper ions to RecA inteins from mycobacterium tuberculosis
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Binding and inhibition of copper ions to RecA inteins from mycobacterium tuberculosis

机译：铜离子与结核分枝杆菌RecA内含肽的结合和抑制

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Protein splicing is a unique post-translational process in which an intein excises itself from a precursor with the concomitant ligation of flank-ing sequences. The binding of zinc to intein inhibits protein splicing reversi-bly and EDTA relieves the inhibition. Copper was found to inhibit protein trans splicing; however, the recovery of intein splicing required both EDTA and TCEP, suggesting a different inhib-ition mechanism for copper compared to zinc. In this work, we have investi-gated the binding properties and inhib-ition effects of copper ions on the RecA intein from Mycobacterium tu - berculosis. Both Cu~+ and Cu~(2+) exhibit-ed high binding affinity to inteins, while different binding sites were iden-tified. Cu~(2+) coordinates to Cys1, the key residue involved in the mechanism of protein splicing, however, Cu~+ does not coordinate to cysteine. An in vitro inhibition assay indicated that monova-lent Cu~+ demonstrates reversible in-hibition to protein splicing, and the in-hibitory efficiency is comparable to Zn ~(2+). Redox reaction between Cu~(2+) and cysteine in inteins were observed and the rate constants were deter-mined. The results suggested a dual role for Cu~(2+) in the inhibition of intein splicing: strong coordination of Cu~(2+) to key residues (including Cys1) in the intein, and subsequent oxidation of Cys1, the residue required for the N→ S acyl shift step in protein splicing. A kinetic study suggested that the coordi-nation could be the major cause of in-hibition effect of Cu~(2+) initially, where-as the redox reaction could play an ad-ditional role in inhibition at a later stage.
机译：蛋白质剪接是独特的翻译后过程，在该过程中，内含肽从前体中切除，伴随着侧翼序列的连接。锌与内含肽的结合可逆地抑制蛋白质剪接，而EDTA可减轻这种抑制。铜被发现可以抑制蛋白质的剪接。然而，恢复内含子剪接需要EDTA和TCEP，这表明与锌相比，铜的抑制机制不同。在这项工作中，我们研究了铜离子对结核分枝杆菌的RecA内含肽的结合特性和抑制作用。 Cu〜+和Cu〜（2+）对内含子均表现出高结合亲和力，而不同的结合位点则被确定。 Cu〜（2+）与Cys1配位，Cys1是参与蛋白质剪接机理的关键残基，而Cu〜+与半胱氨酸不配位。体外抑制试验表明，单价Cu〜+对蛋白质剪接具有可逆的抑制作用，其抑制效率与Zn〜（2+）相当。观察到内含肽中Cu〜（2+）与半胱氨酸之间的氧化还原反应，并确定了速率常数。结果表明，Cu〜（2+）在抑制内含肽剪接中具有双重作用：Cu〜（2+）与内含肽中的关键残基（包括Cys1）有很强的配位作用，随后Cys1的氧化（该残基需要蛋白质剪接中的N→S酰基转移步骤。动力学研究表明，协调可能是Cu〜（2+）最初抑制作用的主要原因，而氧化还原反应则可能在以后的阶段起到抑制作用。

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