P450酶活性中心催化全氟烷基羧酸前体化合物的代谢转化路径研究.pdf

生态毒理学报Asian Journal of Ecotoxicology第 18 卷 第 3 期 2023 年 6 月Vol.18,No.3 Jun.2023 基金项目:国家重点研发计划项目(2022YFC3902105);国家自然科学基金面上项目(22276020);工业生态与环境工程教育部重点实验室开放基金(KLIEEE-21-01)第一作者:常新(1998),女,硕士,研究方向为污染物的生物转化行为模拟预测,E-mail:ChangXinDUT *通信作者(Corresponding author),E-mail:DOI:10.7524/AJE.1673-5897.20230202001常新,于晓梅,傅志强.P450 酶活性中心催化全氟烷基羧酸前体化合物的代谢转化路径研究J.生态毒理学报,2023,18(3):55-67Chang X,Yu X M,Fu Z Q.Investigation on transformation pathways of perfluorinated carboxylic acids precursors catalyzed by active species of P450enzymes J.Asian Journal of Ecotoxicology,2023,18(3):55-67(in Chinese)P450 酶活性中心催化全氟烷基羧酸前体化合物的代谢转化路径研究常新,于晓梅,傅志强*工业生态与环境工程教育部重点实验室,大连市化学品风险防控及污染防治技术重点实验室,大连理工大学环境学院,大连116024收稿日期:2023-02-02 录用日期:2023-04-06摘要:P450 酶催化的前体物质代谢转化是全氟烷基羧酸(PFCAs)重要的间接暴露途径,然而相关反应路径及产物尚不清楚。本研究以多氟烷基磷酸酯(PAP)、氟调聚磺酸(FTS)和氟调聚醇(FTOH)为模型前体,基于密度泛函理论(DFT)计算,揭示了 P450酶活性中心(Cpd)催化 PFCAs 前体的代谢反应机制。DFT 计算发现,3 种前体经 Cpd 催化的烷基 CH 键羟基化及中间体脱烷基重排两步反应,被转化为氟调聚醛(FTAL)中间产物。FTAL 可再次被 Cpd 氧化,经醛基 CH 键羟基化生成氟调聚羧酸(FTCA),与体外实验检测的产物一致。3 种前体与 Cpd 的 CH 反应能垒依次为 PAP FTOH FTS,PAP 反应最容易。对比不同碳链长度的前体发现,PAP 分子链长越短,其与 Cpd 的羟基化反应能垒越低,更容易被转化为 PFCAs。值得注意的是,反应中间体的预测水生急性毒性普遍强于母体及产物。本研究揭示了 PFCAs 前体的 P450 酶促转化路径及产物,有望为 PFCAs 的生物转化归趋、内暴露及健康风险预测奠定科学依据。关键词:全氟烷基羧酸;P450 酶;生物转化;密度泛函理论;间接暴露;前体化合物文章编号:1673-5897(2023)3-055-13 中图分类号:X171.5 文献标识码:AInvestigation on Transformation Pathways of Perfluorinated CarboxylicAcids Precursors Catalyzed by Active Species of P450 EnzymesChang Xin,Yu Xiaomei,Fu Zhiqiang*Key Laboratory of Industrial Ecology and Environmental Engineering(Ministry of Education),Dalian Key Laboratory on ChemicalsRisk Control and Pollution Prevention Technology,School of Environmental Science and Technology,Dalian University of Technology,Dalian 116024,ChinaReceived 2 February 2023 accepted 6 April 2023Abstract:The metabolic conversion of precursors by P450 enzymes represents a significant indirect exposureroute for perfluoroalkyl carboxylic acids(PFCAs)in organisms,whereas the relevant pathways and products remainunknown.In this study,polyfluoroalkyl phosphate(PAP),fluorotelomer sulfonates(FTS),and fluorotelomer alco-hols(FTOH)were used as model precursors to reveal the metabolic mechanism of PFCAs precursors by the activecenter of P450 enzymes(Cpd)using density functional theory(DFT).DFT calculations showed that the three56 生态毒理学报第 18 卷precursors are metabolized by Cpd into a fluorotelomer aldehyde(FTAL)intermediate through a two-step reac-tion involving alkyl CH hydroxylation and dealkylation rearrangement of the hydroxylated intermediates.Sub-sequently,FTAL is further oxidized via formyl CH hydroxylation,leads to the formation of a fluorotelomer car-boxylic acid(FTCA),which is consistent with the experimentally detected product.The CH reaction energybarriers of the three precursors with Cpd followed the order of PAP FTOH 氟调聚醇多氟烷基磷酸酯,表明 PAP 最易被转化成 PFCAs,其次为 FTOH和 FTS。Just 等46研究了 PFCAs 前体在玉米根部的降解速率,发现 PAP 降解速率高于 FTOH,与计算的反应动力学结果一致。2.4 反应产物的生态毒性预测通过 ECOSAR 软件47预测了 PFCAs 前体及P450 转化中间体/产物对鱼的急性毒性(半数致死浓度(LC50),结果如图 8 所示。整体上随碳链增长,中间产物的毒性增强。Mitchell 等48检测了 FTCA 对水藻的毒性,发现 FTCA 毒性随着碳链长度的增加而增加,与 ECOSAR 预测结果一致。此外,对碳链长度相同的 PFCAs 来说,中间体(如 FTAL)毒性显著强于前体化合物及产物。Rand 等49将 PFCAs 中间体代谢物 FTALs、氟调聚物不饱和醛(FTUALs)、FT-CAs 和氟调聚物不饱和羧酸(FTUCAs)与人肝上皮细胞(THLE-2)一起孵育,发现中间体代谢物(FTALs,FTUALs,FTCAs 和 FTUCAs)毒性与官能团有关,强弱顺序为:FTUALs FTALs FTUCAs FTCAs PFCAs,与预测结果一致。因此,在 PFCAs 的风险评估中,代谢中间体及产物的毒性值得关注。但当前体的疏水性较大时(如 82 FTOH 和 82 FTAL 的图 6 有无氢键作用时 Cpd 催化 62 PAP CH 摘氢反应的能量过程图及关键中间物种的优化构型注:X 基团表示C6F13,括号内为四重态值,键长单位为 (0.1 nm),键角单位为(),振动频率为icm-1,能量单位为 kcal mol-1(1 kcal=4.1859 kJ)是酶反应能垒常用的量纲。Fig.6 Optimized geometries of key species and reaction barriers for CH abstraction of 62 PAP catalyzedby Cpd with or without hydrogen bondingNote:The X group represents C6F13,quartet state values are in parentheses,bond distances are in angstroms(,i.e.0.1 nm),angles in degrees(),vibrational frequencies()inicm-1and energy in kcal mol-1(1 kcal=4.1859 kJ)that is the common scale of enzyme reaction energy barriers.64 生态毒理学报第 18 卷辛醇水分配系数 logKow预测值均5),中间体的毒性?反而小于前体,这可能是由于化合物分子量过大,难以跨膜进入生物体,导致其生物积累性及毒性减小。3 讨论(Discussion)基于密度泛函理论计算,阐明了 P450 酶活性中心催化转化典型 PFCAs 前体的反应机制。结果发现 FTAL 是 PFCAs 前体 P450 酶代谢转化过程的共同中间产物。Cpd 先通过 CH 键羟基化将前体转化为醇中间体,而后经非酶环境分子内重排,脱除磺酸基/磷酸基/羟基生成 FTAL,FTAL 进一步与Cpd 发生二次转化生成 FTCAs,相关产物得到体外实验的证实。发现前体化合物分子的磷酸基、磺酸基以及羟基可能通过给电子作用、氢键作用影响反应复合物或过渡态的稳定性,进而改变反应路径和产物。整体上,3 种 PFCAs 前体被 Cpd 转化反应存在动力学差异,反应的难易程度依次为 FTSFTOHPAP。全氟碳链长度越长的 PAP,Cpd 催化转化的能垒越高。PFCAs 前体的生物转化途径复杂,除 P450 酶催化氧化外,还涉及水解、氧化/还原脱氟等过程。例如,DEon 和 Mabury50发现 PAP 在小鼠肠道中可水解生成 FTOH。Martin 等51将分离的大鼠肝细胞与 82 FTOH 一起孵育,检测到了脱氟反应产物82 FTUCA。FTCA 脱氟矿化被认为是其代谢脱毒的重要途径52。因此,PFCAs 前体的其他生物转化途径也值得关注。此外,PFCAs 前体的生物转化是多种生物酶参与的过程。Li 等53的研究表明,氟乙酸脱卤素酶(FAcD)可催化 2-氟丙酸发生 CF 键图 7 不同碳链长度全氟烷基羧酸(PFCAs)前体与化合物反应能垒(1 kcal=4.1859 kJ)Fig.7 Calculated reaction barriers of perfluorinated carboxylicacids(PFCAs)precursors with different fluorocarbonchain lengths catalyzed by Cpd(1 kcal=4.1859 kJ)图 8 PFCAs 前体 P450 酶促转化中间体及产物的鱼水生急性毒性预测结果注:红色区域表示剧毒,橙色区域表示有毒,黄色区域表示有害,绿色区域表示无害。Fig.8 Predicted acute toxicity of fish for transformation intermediates and products of PFCAs precursors by P450sNote:The red region indicates very toxic,the orange region indicates toxic,the yellow region indicatesharmful and the green region indicates not harmful.第 3 期常新等:P450 酶活性中心催化全氟烷基羧酸前体化合物的代谢转化路径研究65 断裂。Peskett 和 Rand54研究发现,人肝脏 S9 以及小肠细胞具有水解酶和氧化酶,使 82 monoPAP 发生转化生成 PFCAs 产物。因此需要研究其他生物酶催化转化 PFCAs 的反应,以全面揭示 PFCAs 的生物转化过程。基于量子化学计算研究 P450 酶催化的 PFCAs前体代谢反应,虽可准确预测转化产物及反应机制,但在实际环境中,不同物种及亚型的 P450 酶活性中心处蛋白环境也存在差异,均可能对反应的动力学产生影响。这方面,量子力学/分子力学(QM/MM)多尺度模拟可以探究真实蛋白环境的影响,已成为研究酶化学反应的重要工具。Lonsdale 等55通过 QM/MM 计算发现双氯芬酸在过渡态时能与 CYP2C9Arg108 侧链形成氢键,导致代谢实验产物与反应优势产物不同。Yadav 等56基于 QM/MM 计算发现,不同亚型 P450 酶因活性中心附近残基差异导致脂肪酸转化产物不同,P450BS通过常规的反弹机制生成羟基化产物,P450OleT通过去羧基化生成烯烃产物。未来考虑使用 QM/MM 方法,以探究 PFCAs 前体在真实酶环境下的转化反应过程及影响因素。获取种类众多 PFASs 物质的毒代动力学参数(如肝脏固有清除率 CLint)是评价其生物积累及毒性的前提。由于计算理论及效率的限制,基于量子力学的方法难以直接计算上述参数。考虑到 P450 酶是生物体内污染物代谢清除的主要酶系,可以在计算不同结构 PFASs 的 P450 酶代谢活化能垒基础上,探索建立毒代动力学参数与反应活性的定量构效关系(QSAR)模型,进而为 PFAS 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