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首页> 外文期刊>Journal of Molecular Biology >Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.
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Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.

机译：来自嗜热脂肪芽孢杆菌的Lysyl-tRNA合成酶：Trp314残基在非极性环境中被屏蔽，并负责氨基酸激活反应中观察到的荧光变化。

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摘要

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.
机译：通过定点诱变产生了来自嗜热脂肪芽孢杆菌的三个赖氨酸-tRNA合成酶的Trp变体，其中酶中的两个Trp残基之一（Trp314和Trp332）被一个Phe残基取代，而没有明显的催化损失。活动。用W332F和野生型酶观察到以下两种现象，而用W314F观察不到：（1）单独添加L-赖氨酸会降低酶的蛋白质荧光，但单独添加ATP不会。 （2）在添加过量的L-赖氨酸之后，随后的ATP的添加使荧光恢复到其原始水平。在各种条件下的荧光测定法和紫外吸收光谱表明，距离赖氨酸结合位点约20A且在非极性环境中被屏蔽的Trp314完全负责L-赖氨酸激活反应中酶的荧光变化。 。此外，尽管L-赖氨酸与溶剂的接触仍然受到限制，但残留物周围的微环境条件在与L-赖氨酸结合时变得更具极性。在比较了荧光发射峰处的波长和相对荧光量子产率之后，建议Trp314的极性比Trp332的极性小。基于某些干扰物的荧光猝灭和N-溴琥珀酰亚胺的化学修饰，认为Trp332位于酶的表面，而Trp314被埋在内部。 L-赖氨酸结合引起的紫外线吸收差异光谱表明，Trp314的状态（包括其静电环境）在此过程中发生了变化，但Trp332并未发生变化。与中性pH相比，在酸性pH下来自Trp314的荧光增强，表明羧酸盐与Trp314残基非常接​​近。

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