Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI 1000, Ljubljana, Slovenia.
Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI 1000, Ljubljana, Slovenia.
Chem Biol Interact. 2023 Sep 1;382:110563. doi: 10.1016/j.cbi.2023.110563. Epub 2023 Jun 5.
Human paraoxonase-1 (PON1) is the most studied member of the paraoxonases (PONs) family and catalyzes the hydrolysis of various substrates (lactones, aryl esters, and paraoxon). Numerous studies link PON1 to oxidative stress-related diseases such as cardiovascular disease, diabetes, HIV infection, autism, Parkinson's, and Alzheimer's, where the kinetic behavior of an enzyme is characterized by initial rates or by modern methods that obtain enzyme kinetic parameters by fitting the computed curves over the entire time-courses of product formation (progress curves). In the analysis of progress curves, the behavior of PON1 during hydrolytically catalyzed turnover cycles is unknown. Hence, progress curves for enzyme-catalyzed hydrolysis of the lactone substrate dihydrocoumarin (DHC) by recombinant PON1 (rePON1) were analyzed to investigate the effect of catalytic DHC turnover on the stability of rePON1. Although rePON1 was significantly inactivated during the catalytic DHC turnover, its activity was not lost due to the product inhibition or spontaneous inactivation of rePON1 in the sample buffers. Examination of the progress curves of DHC hydrolysis by rePON1 led to the conclusion that rePON1 inactivates itself during catalytic DHC turnover hydrolysis. Moreover, human serum albumin or surfactants protected rePON1 from inactivation during this catalytic process, which is significant because the activity of PON1 in clinical samples is measured in the presence of albumin.
人对氧磷酶 1（PON1）是对氧磷酶（PONs）家族中研究最多的成员，能够催化各种底物（内酯、芳基酯和对氧磷）的水解。大量研究将 PON1 与氧化应激相关疾病联系起来，如心血管疾病、糖尿病、HIV 感染、自闭症、帕金森病和阿尔茨海默病，其中酶的动力学行为通过初始速率或现代方法来表征，这些方法通过拟合整个产物形成的时间过程（进展曲线）来获得酶动力学参数。在进展曲线的分析中，未知 PON1 在水解催化周转循环期间的行为。因此，通过重组 PON1（rePON1）对内酯底物二氢香豆素（DHC）的酶促水解进行的进展曲线进行了分析，以研究催化 DHC 周转对 rePON1 稳定性的影响。尽管 rePON1 在催化 DHC 周转过程中明显失活，但由于产物抑制或 rePON1 在样品缓冲液中的自发失活，其活性并未丧失。对 rePON1 催化 DHC 水解的进展曲线的检查得出结论，rePON1 在催化 DHC 水解周转过程中使自身失活。此外，人血清白蛋白或表面活性剂在催化过程中保护 rePON1 不被失活，这一点很重要，因为在白蛋白存在的情况下测量临床样本中的 PON1 活性。
