Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA
Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA
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DOI：
10.1006/jmbi.1996.0527
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发表时间：
1996-10-04
影响因子：
5.6
通讯作者：
Grosjean, H
中科院分区：
文献类型：
--
作者：
Auxilien, S;Crain, PF;Grosjean, H
在酵母中，肌苷存在于七种不同异受体tRNA物种的反密码子的第一位（第34位），而在大肠杆菌中，它仅存在于tRNA（Arg）中。相应的tRNA基因在34位都有腺苷。使用作为底物在体外T7径流成绩单的31质粒携带每个天然的合成tRNA基因窝藏一个反密码子与腺苷34，我们已经表征了酵母酶，催化转换的腺苷34肌苷34。同源E.大肠杆菌酶仅在tRNA中用精氨酸反密码子ACG修饰腺苷34。碱转化通过水解脱氨基型反应发生。这是通过反相高压液相色谱/电喷雾质谱法分析的反应产物后，在[O-18]水中体外修改确定的。这种新的特点tRNA：腺苷34脱氨酶部分纯化酵母。它的分子量约为75 kDa，除了镁离子外，它不需要任何辅助因子来有效地将tRNA中的腺苷34脱氨。所观察到的酶促反应对镁离子的依赖性可能反映了正确的tRNA结构的需要。tRNA的酶识别不依赖于除了腺苷34之外的任何“身份"核苷的存在。同样，反密码子环中假尿苷32或1-甲基-鸟苷37的存在不干扰肌苷34的生物合成。然而，腺苷34转化为肌苷34的效率取决于反密码子环及其近端茎的核苷酸序列，最好的tRNA底物是在35位具有嘌呤的底物。影响反密码子环或几个三维碱基对之一的大小的突变会破坏tRNA作为酵母tRNA：腺苷34脱氨酶底物的能力。显然，酵母tRNA：腺苷34脱氨酶的活性更多地取决于L形tRNA底物的整体结构特征（构象稳定性/灵活性），而不是腺苷34以外的任何特定核苷酸的身份。测得其天然底物tRNA（Ser）（反密码子阿加）的表观Km为2.3 nM。总之，这些结果表明，一个单一的酶可以占肌苷34的存在下，在所有七个细胞质A34的前体tRNA在酵母。(C)1996年学术出版社
In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNA(Arg). The corresponding tRNA genes all have adenosine at position 34. Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34. The homologous E. coli enzyme modifies adenosine 34 only in tRNAs with an arginine anticodon ACG. The base conversion occurs by a hydrolytic deamination-type reaction. This was determined by reversed phase high-pressure liquid chromatography/electrospray mass spectrometry analysis of the reaction product after in vitro modification in [O-18]water. This newly characterised tRNA:adenosine 34 deaminase was partially purified from yeast. It has a molecular mass of approximately 75 kDa, and it does not require any cofactor, except magnesium ions, to deaminate adenosine 34 efficiently in tRNA. The observed dependence of the enzymatic reaction on magnesium ions probably reflects the need for a correct tRNA architecture. Enzymatic recognition of tRNA does not depend on the presence of any ''identity'' nucleoside other than adenosine 34. Likewise, the presence of pseudouridine 32 or 1-methyl-guanosine 37 in the anticodon loop does not interfere with inosine 34 biosynthesis. However, the efficacy of adenosine 34 to inosine 34 conversion depends on the nucleotide sequence of the anticodon loop and its proximal stem, the best tRNA substrates being those with a purine at position 35. Mutations that affect the size of the anticodon loop or one of several three-dimensional base-pairs abolish the capacity of the tRNA to be substrate for the yeast tRNA:adenosine 34 deaminase. Evidently, the activity of yeast tRNA:adenosine 34 deaminase depends more on the global structural feature (conformational stability/flexibility) of the L-shaped tRNA substrates than on the identity of any particular nucleotide other than adenosine 34. An apparent K-m of 2.3 nM for its natural substrate tRNA(Ser) (anticodon AGA) was measured. Altogether, these results suggest that a single enzyme can account for the presence of inosine 34 in all seven cytoplasmic A34-containing precursor tRNAs in yeast. (C) 1996 Academic Press Limited