Marchesani Francesco, Zangelmi Erika, Bruno Stefano, Bettati Stefano, Peracchi Alessio, Campanini Barbara
Department of Food and Drug, University of Parma, 43124 Parma, Italy.
Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43124 Parma, Italy.
Life (Basel). 2021 May 26;11(6):485. doi: 10.3390/life11060485.
Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis-the hydrolysis of phosphoserine to serine and inorganic phosphate-in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through a continuous assay that couples phosphate release to the phosphorolysis of a chromogenic nucleoside by the enzyme purine nucleoside phosphorylase (PNP). These assays suffer from numerous drawbacks, and both rely on the detection of phosphate. We describe a new continuous assay that monitors the release of serine by exploiting bacterial serine acetyltransferase (SAT) as a reporter enzyme. SAT acetylates serine, consuming acetyl-CoA and releasing CoA-SH. CoA-SH spontaneously reacts with Ellman's reagent to produce a chromophore that absorbs light at 412 nm. The catalytic parameters estimated through the SAT-coupled assay are fully consistent with those obtained with the published methods, but the new assay exhibits several advantages. Particularly, it depletes L-serine, thus allowing more prolonged linearity in the kinetics. Moreover, as the SAT-coupled assay does not rely on phosphate detection, it can be used to investigate the inhibitory effect of phosphate on PSP.
磷酸丝氨酸磷酸酶（PSP）催化人类、细菌和植物中从头合成L-丝氨酸的最后一步反应——将磷酸丝氨酸水解为丝氨酸和无机磷酸盐。在已发表的研究中，该反应通常通过间断的孔雀石绿磷酸盐测定法进行监测，或者更罕见地，通过一种连续测定法进行监测，该连续测定法将磷酸盐的释放与酶嘌呤核苷磷酸化酶（PNP）对生色核苷的磷酸解偶联起来。这些测定法存在许多缺点，并且都依赖于磷酸盐的检测。我们描述了一种新的连续测定法，该方法通过利用细菌丝氨酸乙酰转移酶（SAT）作为报告酶来监测丝氨酸的释放。SAT使丝氨酸乙酰化，消耗乙酰辅酶A并释放辅酶A-SH。辅酶A-SH与埃尔曼试剂自发反应生成一种发色团，该发色团在412nm处吸收光。通过SAT偶联测定法估计的催化参数与用已发表方法获得的参数完全一致，但新的测定法具有几个优点。特别是，它消耗L-丝氨酸，从而在动力学上允许更长时间的线性关系。此外，由于SAT偶联测定法不依赖于磷酸盐检测，它可用于研究磷酸盐对PSP的抑制作用。
