Garcia G A, Tierney D L, Chong S, Clark K, Penner-Hahn J E
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, 48109-1065, USA.
Biochemistry. 1996 Mar 5;35(9):3133-9. doi: 10.1021/bi952403v.
A key step in the post-transcriptional modification of tRNA with queuine in Escherichia coli is the exchange of the queuine precursor, preQ1 into tRNA. This reaction is catalyzed by tRNA-guanine transglycosylase (TGT). We have previously shown that the E. coli TGT is a zinc metalloprotein [Chong et al. (1995) Biochemistry 34, 3694-3701]. Site-directed mutagenesis studies indicated that cysteines 302, 304, 307 and histidine 317 constitute the four ligands to the zinc. The involvement of histidine 317 is somewhat confounded by the presence of histidine 316. We have examined the zinc site in TGT (wt) and TGT (H317C) by X-ray absorption spectroscopy. The TGT (wt) data are most consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. Interestingly, the data for TGT (H317C) are also consistent with a tetracoordinate zinc with one nitrogen and three sulfur ligands. The outer shell imidazole scattering for TGT (H317C) appears to be somewhat more ordered than that for TGT (wt), consistent with our previous suggestion that the wild-type enzyme may exist in two conformations the predominant one involving histidine 317 liganding to the zinc and the minor conformer involving histidine 316 liganding to the zinc. The minor conformer, with histidine 316 coordinating the zinc, appears to have an overall conformation that is subtly different from that of the wild-type enzyme. While TGT (H317C) has kinetic parameters very similar to the wild-type, it does not form the homotrimer quaternary structure of the wild-type. TGT (H317A) has previously [Chong et al. (1995) Biochemistry 34, 3694-3701] been found to contain a significant amount of zinc, but is essentially inactive. This suggests that careful analysis of EXAFS data can reveal subtle conformational changes in metal binding sites that are not observed in more common probes of protein conformation such as CD spectroscopy.
在大肠杆菌中，用queuine对tRNA进行转录后修饰的关键步骤是将queuine前体preQ1交换到tRNA中。该反应由tRNA-鸟嘌呤转糖基酶（TGT）催化。我们之前已经表明，大肠杆菌TGT是一种锌金属蛋白[Chong等人（1995年），《生物化学》34卷，3694 - 3701页]。定点诱变研究表明，半胱氨酸302、304、307和组氨酸317构成了锌的四个配体。组氨酸317的参与在某种程度上因组氨酸316的存在而变得复杂。我们通过X射线吸收光谱法研究了TGT（野生型）和TGT（H317C）中的锌位点。TGT（野生型）的数据与具有一个氮和三个硫配体的四配位锌最为一致。有趣的是，TGT（H317C）的数据也与具有一个氮和三个硫配体的四配位锌一致。TGT（H317C）的外壳咪唑散射似乎比TGT（野生型）的更有序，这与我们之前的推测一致，即野生型酶可能以两种构象存在，主要构象涉及组氨酸317与锌配位，次要构象涉及组氨酸316与锌配位。次要构象中组氨酸316与锌配位，其整体构象似乎与野生型酶略有不同。虽然TGT（H317C）的动力学参数与野生型非常相似，但它不会形成野生型的同三聚体四级结构。之前[Chong等人（1995年），《生物化学》34卷，3694 - 3701页]发现TGT（H317A）含有大量锌，但基本上没有活性。这表明对扩展X射线吸收精细结构（EXAFS）数据的仔细分析可以揭示金属结合位点中细微的构象变化，而这些变化在诸如圆二色光谱（CD光谱）等更常见的蛋白质构象探针中是观察不到的。
