Ritter J K, Yeatman M T, Kaiser C, Gridelli B, Owens I S
Section on Genetic Disorders of Drug Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Nov 5;268(31):23573-9.
The characterization (Ritter, J. K., Chen, F., Sheen, Y. Y., Tran, H. M., Kimura, S., Yeatman, M. T., and Owens, I. S. (1992) J. Biol. Chem. 267, 3257-3261) of the single-copy UGT1 gene complex encoding both bilirubin and phenol UDP-glucuronosyltransferases (transferase) has been critical to the determination of genetic defects in Crigler-Najjar Type I patients. The complex (UGT1A-UGT1G) codes for at least two bilirubin, three bilirubin-like, and two phenol transferases. Seven different exons 1, each with an upstream promoter and each encoding the amino terminus of an isoform, are arrayed in series with four common exons (encoding seven identical carboxyl termini) in the 3'-region of the locus. Predictably, a critical mutation in a common exon inactivates the entire locus. A deleterious mutation in an exon 1, as we report here for the UGT1A gene in a Crigler-Najjar Type I patient, predictably affects the amino terminus of that single isoform. The code for the predominant bilirubin isozyme, the HUG-Br1 protein, is missing the phenylalanine codon at position 170 in exon 1 of UGT1A, abolishing a conserved diphenylalanine. We demonstrate that, at the pH (7.6) routinely used for bilirubin glucuronidation studies, both the HUG-Br1 protein and human liver microsomes have approximately one-third the activity seen at the major pH optimum of 6.4 and at low ionic strength. The altered isozyme with nearly normal activity at pH 7.6 is inactive at pH 6.4, a result consistent with the definition of a pH-sensitive mutant. The Km value for bilirubin using the wild-type protein is approximately 2.5 microM at both pH 6.4 and 7.6 and that for the mutant is 5.0 microns at pH 7.6. The structure of the wild-type enzyme compared to that of the mutant indicates that hydrophobic properties at the active center are critical for metabolizing the lipophile-like substrate. The low ion/pH requirements for bilirubin glucuronidation may signal the basis for the distribution of these isozymes to an organelle (endoplasmic reticulum) that can establish compatible conditions/compartments for each catalysis.
编码胆红素和苯酚UDP - 葡萄糖醛酸基转移酶（转移酶）的单拷贝UGT1基因复合体的特性研究（Ritter, J. K., Chen, F., Sheen, Y. Y., Tran, H. M., Kimura, S., Yeatman, M. T., and Owens, I. S. (1992) J. Biol. Chem. 267, 3257 - 3261）对于确定克里格勒 - 纳贾尔I型患者的遗传缺陷至关重要。该复合体（UGT1A - UGT1G）编码至少两种胆红素、三种胆红素样和两种苯酚转移酶。七个不同的外显子1，每个都带有一个上游启动子且各自编码一种同工型的氨基末端，与四个共同外显子（编码七个相同的羧基末端）在该基因座的3'区域串联排列。可以预见，一个共同外显子中的关键突变会使整个基因座失活。正如我们在此报道的克里格勒 - 纳贾尔I型患者中UGT1A基因外显子1中的有害突变，可预见会影响该单一同工型的氨基末端。主要胆红素同工酶HUG - Br1蛋白的编码在UGT1A外显子1的第170位缺失苯丙氨酸密码子，消除了一个保守的双苯丙氨酸。我们证明，在胆红素葡萄糖醛酸化研究常规使用的pH（7.6）条件下，HUG - Br1蛋白和人肝微粒体的活性约为主要最适pH 6.4且离子强度低时活性的三分之一。在pH 7.6时活性接近正常的改变的同工酶在pH 6.4时无活性，这一结果与pH敏感突变体的定义一致。使用野生型蛋白时，胆红素在pH 6.4和7.6时的Km值约为2.5 microM，而突变体在pH 7.6时的Km值为5.0 microM。与突变体相比，野生型酶的结构表明活性中心的疏水特性对于代谢亲脂样底物至关重要。胆红素葡萄糖醛酸化所需的低离子/pH条件可能表明这些同工酶分布到内质网这一细胞器的基础，内质网可为每种催化作用建立合适的条件/区室。
