UDP glucuronosyltransferase (UGT) 1A6 pharmacogenetics: II. Functional impact of the three most common nonsynonymous UGT1A6 Polymorphisms (S7A, T181A, and R184S)
UDP glucuronosyltransferase (UGT) 1A6 pharmacogenetics: II. Functional impact of the three most common nonsynonymous UGT1A6 Polymorphisms (S7A, T181A, and R184S)
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DOI：
10.1124/jpet.104.081968
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发表时间：
2005-06-01
影响因子：
3.5
通讯作者：
Court, MH
中科院分区：
文献类型：
--
作者：
Krishnaswamy, S;Hao, Q;Court, MH
本研究的目的是使用重组酶和人肝微粒体（HLM），以全面评估在人UDP葡萄糖醛酸转移酶（UGT）1A 6基因中发现的三种最常见的非同义多态性（S7 A，T181 A和R184 S）的功能影响。除了已知的等位酶，其他可能的氨基酸变体在人胚肾（HEK）293细胞中表达，以进行结构-功能分析。使用不同底物（5-羟色胺、5-羟基乙醇、4-硝基苯酚、对乙酰氨基酚和丙戊酸）的初步研究显示了相似的结果，与UGT 1A 6 *1（参比品）相比，UGT 1A 6 *2（S7 A/T181 A/R184 S）的葡萄糖醛酸化作用高2倍，其他变体的活性居中。使用UGT 1A 6特异性底物（5-羟色胺）的酶动力学分析显示，与UGT 1A 6 *1相比，所有R184 S变体的Km值降低50%，S7 A/T181 A变体的V-max值升高2倍。此外，UGT 1A 6 *2等位酶的固有清除率（V-max/K-m）值最高（是UGT 1A 6 *1的2.3倍），这是由于更高的酶亲和力和活性的累加效应。正如预期，*1/*1基因分型HLM（5.4 +/- 0.2 mM）的Km值与重组UGT 1A 6 *1（5.8 +/- 0.6 mM）相似。相反，*2/*2 HLM显示较高的Km值（7.0 +/- 0.3 mM），而不是重组UGT 1A 6 *2显示的较低Km值（3.6 +/- 0.3 mM），表明该等位酶可能在HLM中显示与HEK 293细胞不同的酶动力学行为。在最好的情况下，预测这些多态性占观察到的HLM对UGT 1A 6底物葡萄糖醛酸化的13倍变异性的15 - 20%，表明可能存在其他遗传或环境因素导致大多数这种变异。
The objective of this study was to use recombinant enzymes and human liver microsomes (HLMs) to comprehensively evaluate the functional impact of the three most common nonsynonymous polymorphisms (S7A, T181A, and R184S) identified in the human UDP glucuronosyltransferase (UGT) 1A6 gene. In addition to the known allozymes, other possible amino acid variants were expressed in human embryonic kidney (HEK)293 cells to enable structure-function analysis. Initial studies using different substrates (serotonin, 5-hydroxytryptophol, 4-nitrophenol, acetaminophen, and valproic acid) showed similar results with 2-fold higher glucuronidation by UGT1A6*2 (S7A/T181A/R184S) compared with UGT1A6*1 (reference), and intermediate activities for other variants. Enzyme kinetic analyses with the UGT1A6-specific substrate (serotonin) showed 50% lower K m values for all R184S variants and 2-fold higher V-max values for both S7A/T181A variants compared with UGT1A6*1. Furthermore, intrinsic clearance (V-max/K-m) values were highest for the UGT1A6*2 allozyme (2.3-fold over UGT1A6*1), resulting from additive effects of higher enzyme affinity and activity. As expected, K-m values of *1/*1 genotyped HLMs (5.4 +/- 0.2 mM) were similar to recombinant UGT1A6*1 (5.8 +/- 0.6 mM). Conversely, *2/*2 HLMs showed higher K m values (7.0 +/- 0.3 mM) rather than the lower K-m values displayed by recombinant UGT1A6*2 (3.6 +/- 0.3 mM), suggesting that this allozyme may display different enzyme kinetic behavior in HLMs compared with HEK293 cells. At best, these polymorphisms were predicted to account for 15 to 20% of the observed 13-fold variability in glucuronidation of UGT1A6 substrates by HLMs, indicating that there are likely other genetic or environmental factors responsible for the majority of this variation.