Yılmaz Aycan, Dilek Esra
Institute of Science and Technology, Department of Chemistry, Erzincan Binali Yıldırım University, Erzincan, Turkey.
Department of Biochemistry, Division of Pharmaceutical Basic Sciences, Faculty of Pharmacy, Erzincan Binali Yıldırım University, Erzincan 24030, Turkey.
Protein Pept Lett. 2019;26(3):215-220. doi: 10.2174/0929866526666190124144622.
Paraoxonase (PON; arilesterase, [EC 3.1.8.1]) is an enzyme from the group arilesterases (ARE). This enzyme is capable of hydrolyzing paraoxone which is the active metabolite of parathion, an organic phosphorus insecticide. PON activity was found to be low in individuals prone to development of atherosclerosis such as diabetes, familial hypercholesterolemia and kidney disorders. It was noted that PON enzyme activity decreases in relation to age increase in adults. PON enzyme activity is approximately half of that in newborns and premature babies. Approximately one year after birth, it reaches the adult level. It can be said that PON1 has significant role on living organisms. For this reason, many studies on interactions of PON-drugs are needed.
In this article, our aim is to investigate in vitro effects of four pharmaceutically active agents (fosfomycin, cefuroxime axetil, cefaclor monohydrate, and cefixime) which are often used in patients after surgery on human serum paraoxanase-I (PON1) enzyme activity.
In this article, we purify paraoxonase-I enzyme from human serum by using ammonium sulfate precipitation (in the range of 60-80%), ion exchange and gel filtration chromatography. We use electrophoresis to check the purity of the enzyme. We investigate the paraoxonase activity of the enzyme at 412 nm the inhibition effects of the active substances. Paraoxone is used as the substrate. Activity measurements arw made at different inhibitor concentrations related to inhibitor studies and % Activity- [I] graphs are drawn for drug active substances. Lineweaver-Burk graphics are used to determine the Ki constants. Finally, to determine the types of inhibition we interpret these graphs.
The active agents used after surgery decreased the PON1 enzyme activity. They showed different inhibition mechanism. The inhibition mechanism of fosfomycin and cefaclor monohydrate was noncompetitive, cefixime was uncompetitive and cefuroxime axetil was a competitive inhibitor. The IC50 values for fosfomycin, cefuroxime axetil, cefaclor monohydrate, and cefixime were calculated to be 31.5 mM, 1.03 mM, 4.18 mM and 0.781 mM, respectively, and the Ki constants were determined to be 27.98 ± 12.25 mM, 2.20 ± 0.22 mM, 4.81 ± 2.25 mM and 1.12 ± 0.32 mM, respectively. The IC50 and Ki values showed that cefixime active agent has the maximum inhibition.
In this study, we have detected that cefuroxime axetil inhibited competitively in vitro paraoxonase activity of this enzyme. According to this information, we thought that cefuroxime axetil linked to the active site of the enzyme. Fosfomycin and cefaclor monohydrate can be attached with amino acids out of the active site of the enzyme because they inhibit enzyme noncompetitively. Cefixime can be attached only to the enzyme-substrate complex because it inhibits enzyme uncompetitively.
对氧磷酶（PON；芳基酯酶，[EC 3.1.8.1]）是芳基酯酶（ARE）家族中的一种酶。这种酶能够水解对氧磷，对氧磷是有机磷杀虫剂对硫磷的活性代谢产物。在易患动脉粥样硬化的个体如糖尿病患者、家族性高胆固醇血症患者和肾脏疾病患者中，发现PON活性较低。值得注意的是，在成年人中，PON酶活性随年龄增长而降低。PON酶活性约为新生儿和早产儿的一半。出生后约一年，其达到成人水平。可以说，PON1对生物体具有重要作用。因此，需要进行许多关于PON与药物相互作用的研究。
在本文中，我们的目的是研究四种常用于术后患者的药物活性剂（磷霉素、头孢呋辛酯、头孢克洛一水合物和头孢克肟）对人血清对氧磷酶-I（PON1）酶活性的体外影响。
在本文中，我们通过硫酸铵沉淀（60%-80%范围内）、离子交换和凝胶过滤色谱从人血清中纯化对氧磷酶-I酶。我们使用电泳检查酶的纯度。我们在412nm处研究该酶的对氧磷酶活性以及活性物质的抑制作用。以对氧磷作为底物。在与抑制剂研究相关的不同抑制剂浓度下进行活性测量，并绘制药物活性物质的%活性-[I]图。使用Lineweaver-Burk图来确定Ki常数。最后，为了确定抑制类型，我们解读这些图。
术后使用的活性剂降低了PON1酶活性。它们表现出不同的抑制机制。磷霉素和头孢克洛一水合物的抑制机制是非竞争性的，头孢克肟是反竞争性的，头孢呋辛酯是竞争性抑制剂。磷霉素、头孢呋辛酯、头孢克洛一水合物和头孢克肟的IC50值分别计算为31.5mM、1.03mM、4.18mM和0.781mM，Ki常数分别确定为27.98±12.25mM、2.20±0.22mM、4.81±2.25mM和1.12±0.32mM。IC50和Ki值表明头孢克肟活性剂具有最大抑制作用。
在本研究中，我们检测到头孢呋辛酯在体外竞争性抑制该酶的对氧磷酶活性。根据这一信息，我们认为头孢呋辛酯与酶的活性位点相连。磷霉素和头孢克洛一水合物可以与酶活性位点之外的氨基酸结合，因为它们非竞争性抑制酶。头孢克肟只能与酶-底物复合物结合，因为它反竞争性抑制酶。
