Glasfeld E, Landro J A, Schimmel P
Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Biochemistry. 1996 Apr 2;35(13):4139-45. doi: 10.1021/bi9527810.
Escherichia coli isoleucyl-tRNA synthetase is one of five closely related class I tRNA synthetases. The active site of the 939 amino acid polypeptide is in an N-terminal domain which contains an insertion believed essential for interactions with the tRNA acceptor helix. The enzyme was shown previously to contain an essential (for function in vivo) zinc bound to a Cys4 cluster at the C-terminal end of the polypeptide. The specific function of this zinc has been unknown. We show here that aminoacylation activity can be reconstituted in vitro by combining a 53 amino acid zinc-containing C-terminal peptide with a protein consisting of the remaining 886 amino acids. Reconstitution of aminoacylation is zinc-dependent. In contrast, the zinc-containing peptide is dispensable for synthesis of isoleucyl adenylate. Affinity coelectrophoresis showed that the 53 amino acid C-terminal peptide is required specifically for tRNA binding. We propose that the zinc-containing peptide curls back to the active site to make contact with the acceptor helix of bound tRNA, but not with isoleucine or ATP. It is the first example of a zinc-containing peptide in a class I tRNA synthetase that is essential for tRNA binding interactions. The design of this enzyme may be part of a more general scheme for class I tRNA synthetases to acquire acceptor helix binding elements during the development of the genetic code.
大肠杆菌异亮氨酰 - tRNA合成酶是五种密切相关的I类tRNA合成酶之一。这个由939个氨基酸组成的多肽的活性位点位于N端结构域，该结构域包含一个插入序列，据信该插入序列对于与tRNA受体螺旋的相互作用至关重要。先前已表明该酶含有一个必需的（对于体内功能而言）锌，该锌与多肽C端的一个Cys4簇结合。这种锌的具体功能一直未知。我们在此表明，通过将一个含53个氨基酸的含锌C端肽与由其余886个氨基酸组成的蛋白质相结合，可以在体外重建氨基酰化活性。氨基酰化的重建是锌依赖性的。相比之下，含锌肽对于异亮氨酰腺苷酸的合成是可有可无的。亲和共电泳表明，这个含53个氨基酸的C端肽是tRNA结合所特需的。我们提出，含锌肽卷曲回到活性位点，以便与结合的tRNA的受体螺旋接触，但不与异亮氨酸或ATP接触。这是I类tRNA合成酶中一个含锌肽的首个例子，该肽对于tRNA结合相互作用至关重要。这种酶的设计可能是I类tRNA合成酶在遗传密码发展过程中获取受体螺旋结合元件的更普遍方案的一部分。
