Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1
Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1
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DOI：
10.1074/jbc.m009341200
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发表时间：
2001-05-04
影响因子：
4.8
通讯作者：
Tanguay, RM
中科院分区：
文献类型：
--
作者：
Bergeron, A;D'Astous, M;Tanguay, RM
遗传性酪氨酸血症1型（HT1）是一种常染色体隐性遗传疾病，由酪氨酸降解的最后一步酶富马酰乙酰乙酸水解酶（FAH）缺乏引起。迄今为止，在各种HT1患者中已报道了34种FAH基因突变。用定点突变法研究了HTI患者中发现的8个FAH错义突变对FAH结构和活性的影响。突变N16 I、F62 C、A134 D、C193 R、D233 V和W234 G导致无酶活性的FAH蛋白。两个突变（R341W，与假缺陷表型，和Q279R）产生的蛋白质的活性水平与野生型酶相当。N16 I、F62 C、C193 R和W234 G变体在不溶性细胞级分中富集，表明这些氨基酸取代干扰酶的正确折叠。基于FAH的三级结构，圆二色谱数据，和溶解度测量，我们提出，研究的错义突变导致三种类型的酶的结构影响：1）总的结构扰动，2）有限的构象变化的活性位点，和3）构象修饰，对酶活性没有显着的影响。
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by a deficiency of the enzyme involved in the last step of tyrosine degradation, fumarylacetoacetate hydrolase (FAH), Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagenesis of the FAH cDNA was used to investigate the effects of eight missense mutations found in HTI patients on the structure and activity of FAH, Mutated FAH proteins were expressed in Escherichia coli and in mammalian CV 1 cells. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatically inactive FAH proteins. Two mutations (R341W, associated with the pseudo-deficiency phenotype, and Q279R) produced proteins with a level of activity comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G variants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cause three types of structural effects on the enzyme: 1) gross structural perturbations, 2) limited conformational changes in the active site, and 3) conformational modifications with no significant effect on enzymatic activity.