Differential regulation of opposing RelMtb activities by the aminoacylation state of a tRNA•ribosome•mRNA•RelMtb complex
Differential regulation of opposing RelMtb activities by the aminoacylation state of a tRNA•ribosome•mRNA•RelMtb complex
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DOI：
10.1021/bi001256k
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发表时间：
2000-09-26
期刊：
影响因子：
2.9
通讯作者：
Rubin, H
中科院分区：
文献类型：
--
作者：
Avarbock, D;Avarbock, A;Rubin, H
结核分枝杆菌的Rel（Mtb）负责（p）ppGpp的细胞内调节以及随后生物体在长期饥饿中存活的能力，表明在结核病的发病机制中可能起作用。纯化的Rel（Mtb）是进行ATP：GTP/GDP/ITP 3 '-焦磷酸转移酶和（p）ppGpp 3'-焦磷酸水解酶反应的双功能酶。在这里，我们表明，在没有生物调节剂的情况下，Rel（Mtb）同时催化每个反应的最大速率的转移酶和水解，表明存在两个不同的活性位点，Rel（Mtb）的相反活性的差异调节依赖于不带电的tRNA与带电的tRNA的比率以及Rel（Mtb）与包含tRNA、核糖体和mRNA的复合物的缔合，当RelMtb与含有不带电荷的tRNA、核糖体和mRNA的复合物（Rel（Mtb）激活复合物或RAG）结合时，k（cat）从转移酶活性的基础水平增加20倍，K-ATP和K-GTP降低4倍。然而，在Rel（Mtb）活化复合物的存在下，水解的k（cat）从基础水平降低2倍，pppGpp的K-m从基础水平增加2倍。向该复合物中加入带电荷的tRNA通过抑制转移酶活性和激活水解活性而具有相反的效果。RelMtb的差异控制使Mtb核糖体复合物在响应于可能存在于休眠Mtb病变中的严格生长条件而控制细胞代谢中具有新的调节作用。
Rel(Mtb) of Mycobacterium tuberculosis is responsible for the intracellular regulation of (p)ppGpp and the consequent ability of the organism to survive long-term starvation, indicating a possible role in the pathogenesis of tuberculosis. Purified Rel(Mtb) is a dual-function enzyme carrying out ATP: GTP/GDP/ITP 3'-pyrophosphoryltransferase and (p)ppGpp 3'-pyrophosphohydrolase reactions. Here we show that in the absence of biological regulators, Rel(Mtb) simultaneously catalyzes both transferase and hydrolysis at the maximal rate for each reaction, indicating the existence of two distinct active sites, The differential regulation of the opposing activities of Rel(Mtb) is dependent on the ratio of uncharged to charged tRNA and the association of Rel(Mtb) with a complex containing tRNA, ribosomes, and mRNA, A 20-fold increase in the k(cat) and a 4-fold decrease in K-ATP and K-GTP from basal levels for transferase activity occur when RelMtb binds to a complex containing uncharged tRNA, ribosomes, and mRNA (Rel(Mtb) activating complex or RAG). The k(cat) for hydrolysis, however, is reduced 2-fold and K-m for pppGpp increased 2-fold from basal levels in the presence of the Rel(Mtb) activating complex. The addition of charged tRNA to this complex has the opposite effect by inhibiting transferase activity and activating hydrolysis activity. Differential control of RelMtb gives the Mtb ribosomal complex a new regulatory role in controlling cellular metabolism in response to stringent growth conditions that may be present in the dormant Mtb lesion.