Fisher Gemma, Pečaver Ennio, Read Benjamin J, Leese Susannah K, Laing Erin, Dickson Alison L, Czekster Clarissa M, da Silva Rafael G
School of Biology, University of St Andrews, Biomedical Sciences Research Complex, St Andrews, Fife KY16 9ST, U.K.
ACS Catal. 2023 May 23;13(11):7669-7679. doi: 10.1021/acscatal.3c01111. eCollection 2023 Jun 2.
The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of -(5-phospho-β-D-ribosyl)-ATP (PRATP) to -(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to -(5'-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV-VIS spectroscopy and LC-MS to show putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on and / ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized -(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel.
双功能酶磷酸核糖基 - ATP焦磷酸水解酶/磷酸核糖基 - AMP环水解酶（HisIE）催化组氨酸生物合成的第二步和第三步反应：在C端HisE样结构域中，将β-(5 - 磷酸 - β - D - 核糖基)-ATP（PRATP）焦磷酸水解为β-(5 - 磷酸 - β - D - 核糖基)-AMP（PRAMP）和焦磷酸；在N端HisI样结构域中，将PRAMP环水解为β-(5'-磷酸 - D - 核糖基甲亚胺基)-5 - 氨基 - 1-(5″-磷酸 - D - 核糖基)-4 - 咪唑甲酰胺（ProFAR）。在此，我们利用紫外 - 可见光谱法和液相色谱 - 质谱联用技术表明，假定的HisIE能从PRATP生成ProFAR。通过一种检测焦磷酸的测定法和另一种检测ProFAR的测定法，我们确定焦磷酸水解酶反应速率高于总反应速率。我们制备了该酶的截短版本，仅包含C端（HisE）结构域。这种截短的HisIE具有催化活性，能够合成环水解反应的底物PRAMP。PRAMP在动力学上适合HisIE催化生成ProFAR，这表明PRAMP可以从大量水中结合到HisI样结构域，并且表明环水解反应是整个双功能酶的限速步骤。总反应速率随着pH值升高而增加，而溶剂氘动力学同位素效应在碱性更强的pH值下降低，但在pH 7.5时仍然很大。溶剂粘度对反应速率和产物释放速率没有影响，排除了扩散步骤限制底物结合和产物释放速率的可能性。使用过量PRATP的快速动力学研究表明，在ProFAR形成过程中存在一个滞后时间，随后是一个爆发阶段。这些观察结果与一个限速单分子步骤一致，该步骤涉及腺嘌呤环打开后的质子转移。我们合成了β-(5 - 磷酸 - β - D - 核糖基)-ADP（PRADP），它不能被HisIE处理。PRADP抑制HisIE催化从PRATP生成ProFAR，但不抑制从PRAMP生成ProFAR，这表明它结合到磷酸水解酶活性位点，同时仍然允许PRAMP无障碍地进入环水解酶活性位点。动力学数据与PRAMP在大量溶剂中的积累不相符，表明HisIE催化涉及PRAMP的优先通道化，尽管不是通过蛋白质通道。
