Research Group Inborn Errors of Metabolism, Department of Natural Sciences,, Bonn-Rhein-Sieg University of Applied Sciences, von-Liebig-Str. 20, 53359, Rheinbach, Germany.
Division of Clinical Chemistry & Biochemistry, University Children's Hospital and Children's Research Center, Zürich, Switzerland.
Metab Brain Dis. 2019 Dec;34(6):1629-1634. doi: 10.1007/s11011-019-00470-9. Epub 2019 Jul 30.
Acylpeptide hydrolase (APEH) is a serine protease involved in the recycling of amino acids from acylated peptides. Beyond that, APEH participates in the metabolism of the antiepileptic drug valproic acid (2-propylpentanoic acid; VPA) by catalyzing the hydrolysis of the VPA metabolite valproylglucuronide (VPA-G) to its aglycon. It has been shown that the inhibition of APEH by carbapenem antibiotics decreases therapeutic VPA levels by enhancing the urinary elimination of VPA in form of VPA-G. As various sequence variants of the APEH gene (which encodes the APEH protein) are listed in databases, but have not been functionally characterized yet, we assume, that some APEH sequence variants may have pharmacogenetic relevance due to their impaired cleavage of VPA-G. APEH sequence variants predicted to affect enzyme activity were selected from databases, and overexpressed in HEK293 cells (stable transfection), a cell line derived from human embryonic kidney cells. APEH activity in cell homogenates was determined spectrophotometrically by monitoring the hydrolysis of the synthetic substrate N-acetyl-L-alanine-nitroanilide. APEH enzyme activity and protein expression of the sequence variants were compared with those of APEH with the reference sequence. Three out of five tested missense sequence variants resulted in a considerable decrease of enzyme activity assessed with the standard substrate N-acetyl-L-alanine-nitroanilide, suggesting an effect on pharmacokinetics of VPA. Our work underlines the need to consider the APEH genotype in investigations of altered VPA metabolism.
酰肽水解酶 (APEH) 是一种丝氨酸蛋白酶，参与从酰化肽中回收氨基酸。除此之外，APEH 通过催化 VPA 代谢物丙戊酰葡糖醛酸 (VPA-G) 水解为其苷元，参与抗癫痫药物丙戊酸 (2-丙基戊酸；VPA) 的代谢。已经表明，碳青霉烯类抗生素抑制 APEH 通过以 VPA-G 的形式增强 VPA 的尿消除来降低治疗性 VPA 水平。由于 APEH 基因（编码 APEH 蛋白）的各种序列变异体已在数据库中列出，但尚未进行功能表征，因此我们假设，由于其对 VPA-G 的切割受损，一些 APEH 序列变异体可能具有药物遗传学相关性。从数据库中选择了预测会影响酶活性的 APEH 序列变异体，并在 HEK293 细胞（稳定转染）中过表达，HEK293 细胞系源自人胚肾细胞。通过监测合成底物 N-乙酰-L-丙氨酸-硝基苯胺的水解，分光光度法测定细胞匀浆中的 APEH 活性。将序列变异体的 APEH 酶活性和蛋白表达与参考序列的 APEH 进行比较。在所测试的五个错义序列变异体中有三个导致用标准底物 N-乙酰-L-丙氨酸-硝基苯胺评估的酶活性显着降低，这表明对 VPA 药代动力学有影响。我们的工作强调了在研究改变的 VPA 代谢时需要考虑 APEH 基因型。
