Parkins Andrew, Sandin Suzanne I, Knittel Jonathon, Franz Andreas H, Ren Jianhua, de Alba Eva, Pantouris Georgios
Department of Chemistry, University of the Pacific, Stockton, California 95211, United States.
Department of Bioengineering, University of California, Merced, California 95343, United States.
Anal Chem. 2023 Mar 21;95(11):4957-4965. doi: 10.1021/acs.analchem.2c04969. Epub 2023 Mar 6.
Macrophage migration inhibitory factor (MIF) is a key immunostimulatory protein with regulatory properties in several disorders, including inflammation and cancer. All the reported inhibitors that target the biological activities of MIF have been discovered by testing against its keto/enol tautomerase activity. While the natural substrate is still unknown, model MIF substrates are used for kinetic experiments. The most extensively used model substrate is 4-hydroxyphenyl pyruvate (4-HPP), a naturally occurring intermediate of tyrosine metabolism. Here, we examine the impact of 4-HPP impurities in the precise and reproducible determination of MIF kinetic data. To provide unbiased evaluation, we utilized 4-HPP powders from five different manufacturers. Biochemical and biophysical analyses showed that the enzymatic activity of MIF is highly influenced by underrepresented impurities found in 4-HPP. Besides providing inconsistent turnover results, the 4-HPP impurities also influence the accurate calculation of ISO-1's inhibition constant, an MIF inhibitor that is broadly used for and studies. The macromolecular NMR data show that 4-HPP samples from different manufacturers result in differential chemical shift perturbations of amino acids in MIF's active site. Our MIF-based conclusions were independently evaluated and confirmed by 4-hydroxyphenylpyruvate dioxygenase (HPPD) and D-dopachrome tautomerase (D-DT); two additional enzymes that utilize 4-HPP as a substrate. Collectively, these results explain inconsistencies in previously reported inhibition values, highlight the effect of impurities on the accurate determination of kinetic parameters, and serve as a tool for designing error-free and experiments.
巨噬细胞移动抑制因子（MIF）是一种关键的免疫刺激蛋白，在包括炎症和癌症在内的多种疾病中具有调节特性。所有已报道的针对MIF生物活性的抑制剂都是通过测试其酮/烯醇互变异构酶活性发现的。虽然天然底物仍不清楚，但模型MIF底物用于动力学实验。使用最广泛的模型底物是4-羟基苯丙酮酸（4-HPP），它是酪氨酸代谢的天然中间产物。在此，我们研究了4-HPP杂质对精确且可重复测定MIF动力学数据的影响。为了提供无偏评估，我们使用了来自五个不同制造商的4-HPP粉末。生化和生物物理分析表明，MIF的酶活性受到4-HPP中含量不足的杂质的高度影响。除了提供不一致的周转结果外，4-HPP杂质还影响ISO-1抑制常数的准确计算，ISO-1是一种广泛用于相关研究的MIF抑制剂。大分子核磁共振数据表明，来自不同制造商的4-HPP样品会导致MIF活性位点氨基酸的化学位移扰动不同。我们基于MIF得出的结论由4-羟基苯丙酮酸双加氧酶（HPPD）和D-多巴色素互变异构酶（D-DT）独立评估和确认；这两种额外的酶也将4-HPP用作底物。总体而言，这些结果解释了先前报道的抑制值的不一致性，突出了杂质对动力学参数准确测定的影响，并可作为设计无误差实验的工具。
