TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE FROM ESCHERICHIA-COLI - STRUCTURE-ACTIVITY STUDIES INVESTIGATING THE ROLE OF THE AMINOMETHYL SUBSTITUENT OF THE HETEROCYCLIC SUBSTRATE PREQ(1)
TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE FROM ESCHERICHIA-COLI - STRUCTURE-ACTIVITY STUDIES INVESTIGATING THE ROLE OF THE AMINOMETHYL SUBSTITUENT OF THE HETEROCYCLIC SUBSTRATE PREQ(1)
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DOI：
10.1021/bi00046a047
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发表时间：
1995-11-21
期刊：
影响因子：
2.9
通讯作者：
GARCIA, GA
中科院分区：
文献类型：
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作者：
HOOPS, GC;TOWNSEND, LB;GARCIA, GA
为了研究大肠杆菌 tRNA-鸟嘌呤转糖基酶 (TGT) 的底物特异性，合成了一系列 5-取代 2-氨基吡咯并[2,3-d]嘧啶-4(3H)-酮。许多这些化合物最初被检查为 TGT 催化的放射性标记鸟嘌呤掺入 tRNA 的抑制剂 [Hoops, G. C., Garcia, G. A., & Townsend, L. B. (1992) 第 204 届美国化学会全国会议，华盛顿特区，1992 年 8 月 23-28 日，药物化学部，摘要 113]。通过监测 8-[C-14]G34-tRNA 中放射性标记鸟嘌呤的损失，确定了这些类似物作为 TGT 反应底物的动力学参数。这项研究表明，来自大肠杆菌的 tRNA-鸟嘌呤转糖基酶能够耐受 5 位上的多种取代基。 5-取代基的作用似乎完全在于结合/识别，对催化没有明显影响。 N7 pK(a) 和 V-max 之间的相关性表明，反应过程中 N7 的去质子化必须在随后的糖苷键形成之前发生，似乎是天然底物的部分速率决定因素。 7-甲基取代的竞争性抑制剂的 K(i) 与其相应底物的 K(m) 的比较表明，某些底物（包括 preQ(1)）在动力学上具有“粘性”（即 K-m 相当于 K-d），而其他底物的 K(m) 反映了催化速率以及结合。
A series of 5-substituted 2-aminopyrrolo[2,3-d]pyrimidin-4(3H)-ones have been synthesized in order to study the substrate specificity of the tRNA-guanine transglycosylase (TGT) from Escherichia coli. A number of these compounds were initially examined as inhibitors of radiolabeled guanine incorporation into tRNA catalyzed by TGT [Hoops, G. C., Garcia, G. A., & Townsend, L. B. (1992) 204th National Meeting of the American Chemical Society, Washington, DC, August 23-28, 1992, Division of Medicinal Chemistry, Abstract 113]. The kinetic parameters of these analogues as substrates in the TGT reaction have been determined by monitoring the loss of radiolabeled guanine from 8-[C-14]G34-tRNA. This study reveals that the tRNA-guanine transglycosylase from E. coli will tolerate a wide variety of substituents at the 5-position. The role of the 5-substituent appears to be entirely in binding/recognition with no apparent effects upon catalysis. A correlation between N7 pK(a) and V-max suggests the deprotonation of N7 during the reaction, which must occur prior to subsequent glycosidic bond formation, appears to be partially rate-determining for the natural substrate. Comparison of the K(i)s of 7-methyl-substituted competitive inhibitors to the K(m)s of their corresponding substrates suggests that some substrates (including preQ(1)) are kinetically ''sticky'' (i.e., K-m is equivalent to K-d) and other substrates have K(m)s that reflect catalytic rates as well as binding.