Finding Ways to Relax: A Revisionistic Analysis of the Chemistry of E. coli GTP Cyclohydrolase II
Finding Ways to Relax: A Revisionistic Analysis of the Chemistry of E. coli GTP Cyclohydrolase II
复制标题
寻找放松的方法：大肠杆菌 GTP 环水解酶 II 化学的修正分析
DOI：
10.1021/acs.biochem.1c00511
复制
发表时间：
2021
期刊：
影响因子：
2.9
通讯作者：
Moran, Graham R.
中科院分区：
文献类型：
--
作者：
Smith, Madison M.;Beaupre, Brett A.;Fourozesh, Dariush C.;Meneely, Kathleen M.;Lamb, Audrey L.;Moran, Graham R.
三磷酸鸟苷 (GTP) 环水解酶 II (RibA) 是水解裂解 GTP 鸟嘌呤 C8-N9 键的三种酶之一。 RibA 还催化随后的碱基水解攻击，释放甲酸盐，此外还裂解三磷酸盐的 α-β 磷酸二酯键，形成焦磷酸盐 (PPi)。这些水解反应由串联活性位点金属离子锌和镁促进，它们分别在 GTP 鸟嘌呤和三磷酸部分发挥作用。 RibA 反应是核黄素生物合成的一部分，形成 2,5-二氨基-6-β-嘧啶酮 5'-磷酸，这是一种环外嘧啶核苷酸，最终形成核黄素异咯嗪的嘧啶环。 RibA 反应的化学计量在首次在大肠杆菌中鉴定出这种活性的研究中被定义（Foor, F., Brown, G. M.J. Biol. Chem., 1975, 250, 9, 3545−3551），但在后续工作中尚未进行定量评估。我们主要使用瞬态方法检查了来自 E 的 RibA 的相互作用。大肠杆菌与 GTP、肌苷三磷酸和 PPi。我们的数据表明，PPi 是 RibA 的缓慢底物，被裂解形成两个磷酸根离子 (Pi)。实时酶联 Pi 报告基因检测和终点 31 P NMR 相结合表明，Pi 在 RibA 与 GTP 的天然反应中以催化相关速率形成，重新定义了反应化学计量。此外，我们的数据表明，PPi 和 GTP 在水解化学之前都会刺激构象变化，我们得出结论，作为底物或中间态结合的 PPi 的裂解会导致构象松弛。
Guanosine triphosphate (GTP) cyclohydrolase II (RibA) is one of three enzymes that hydrolytically cleave the C8–N9 bond of the GTP guanine. RibA also catalyzes a subsequent hydrolytic attack at the base liberating formate and in addition cleaves the α–β phosphodiester bond of the triphosphate to form pyrophosphate (PPi). These hydrolytic reactions are promoted by tandem active-site metal ions, zinc and magnesium, that respectively function at the GTP guanine and triphosphate moieties. The RibA reaction is part of riboflavin biosynthesis and forms 2,5-diamino-6-β-pyrimidinone 5′-phosphate, an exocyclic pyrimidine nucleotide that ultimately forms the pyrimidine ring of the isoalloxazine of riboflavin. The stoichiometry of the RibA reaction was defined in the study that first identified this activity inEscherichia coli(Foor, F., Brown, G. M.J. Biol. Chem., 1975, 250, 9, 3545−3551) and has not been quantitatively evaluated in subsequent works. Using primarily transient state approaches we examined the interaction of RibA fromE. coliwith the GTP, inosine triphosphate, and PPi. Our data indicate that PPi is a slow substrate for RibA that is cleaved to form two phosphate ions (Pi). A combination of real-time enzymatically coupled Pi reporter assays and end-point31P NMR revealed that Pi is formed at a catalytically relevant rate in the native reaction of RibA with GTP, redefining the reaction stoichiometry. Furthermore, our data indicate that both PPi and GTP stimulate conformational changes prior to hydrolytic chemistry, and we conclude that the cleavage of PPi bound as a substrate or an intermediate state results in conformational relaxation.