Toth M J, Schimmel P
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1990 Jan 15;265(2):1005-9.
A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.
设计了一种策略来分离在氨基酸结合位点发生改变的甘氨酰 - tRNA合成酶突变体，包括一类氨基酸特异性改变的突变体。为此，质粒pBR322发生突变，使得β - 内酰胺酶基因中活性位点Ser - 68的密码子（AGC）变为甘氨酸密码子GGC，从而使编码的酶失活。分离出了能增加β - 内酰胺酶的Gly - 68等位基因的β - 内酰胺酶活性的抑制子，其中一些被定位到编码甘氨酰 - tRNA合成酶（glyS）的基因上。虽然未检测到任何突变体对tRNA(Gly)进行丝氨酸的体外错氨基酰化，但甘氨酰 - tRNA合成酶活性发生了改变。对一个严重受影响的glyS突变体（N302）进行了更详细的研究。对于这个突变体，α链中单个Pro - 61→Leu取代导致氨基酰化反应中甘氨酸的Km值升高（25倍），ATP的Km值升高（45倍），但对tRNA的Km值只有轻微扰动。腺苷酸合成活性也严重降低（超过100倍）。此外，观察到氨基酰化活性与酶浓度之间存在非线性关系，这意味着α链Pro - 61→Leu突变破坏了全酶功能上必需的亚基相互作用。前文的结果表明，α链和部分β链对于氨基酰化和腺苷酸合成活性是必需的。本研究结果表明，α链以一种受适当亚基相互作用影响的方式，对氨基酸和ATP结合有特定贡献。
