苯丙氨酸羟化酶基因G247S、E280G、P362T和A434D突变的体外表达研究和结构分析  

In vitro expression and structural analysis of four missense mutations （G247S, E280G, P362T, A434D） of phenylalanine hydroxylase gene

作　　者：宋昉[1] 瞿宇晋[1] Yoshiyuki Okano 叶志强[3] 张玉敏 金煜炜[1] 王红[1] 

机构地区：[1]首都儿科研究所医学遗传研究室,北京100020 [2]Department of Pediatrics, Osaka City University Graduate School of Medicine,Osaka, Japan [3]北京大学生命科学学院生物信息中心 [4]北京市新生儿疾病筛查中心

出　　处：《中华医学遗传学杂志》2008年第1期1-5,共5页Chinese Journal of Medical Genetics

基　　金：北京市自然科学基金（5052008）

摘　　要：目的探讨苯丙氨酸羟化酶基因4种新错义突变G247S、E280G、P362T和A434D的致病性质以及与临床表型之间的关系。方法（1）应用体外真核表达体系进行突变体蛋白的瞬时表达；以野生型苯丙氨酸羟化酶（phenylalanine hydroxide，PAH）的酶活性作为参照，计算突变体的残余酶活性。（2）比对不同种属的PAH酶蛋白序列，分析这些突变位点的氨基酸的保守性。（3）以正常PAH蛋白的晶体结构为基础，应用生物大分子结构模拟软件进行突变位点的三维结构分析，预测氨基酸的替换对酶功能的潜在影响。（4）根据治疗前血苯丙氨酸浓度和饮食苯丙氨酸的耐受量，进行苯丙酮尿症（phenylketonuria，PKU）患儿的临床表型分析。结果（1）突变体G247S、E280G、P362T和A434D体外表达的残余酶活性分别为野生型的3．1％、0．4％、8．2％和21．7％。（2）Gly247、G1u280、Pr0362均属于高度保守的氨基酸残基位点，而Ala434则属于比较保守的位点。（3）三维分子结构分析显示G2A,7S和E280G位于酶的活性中心位置，可能分别影响PAH酶与四氢生物蝶呤和铁离子的结合；而突变P362T和A434D则可能分别影响PAH酶二聚体和四聚体化的形成和稳定性。（4）临床表型分析显示携带G247S和E280G突变的患者为经典型PKU，携带P362T的患者既有经典型PKU也有中间型PKU，携带A434D患者均为中间型PKU。结论（1）E280G、G247S、P362T和A434D突变均为致病性的突变，位于酶活性中心位点的突变影响酶的活性功能更为明显。（2）突变酶分子的结构分析以及对酶功能影响的预测与体外表达的实验结果基本一致。（3）突变基因型、体外表达活性实验以及临床表型之间的关联分析存在着一定的关联性。Objective To understand the pathogenic effect and the correlation between the genotype and phenotype of the 4 novel missense mutations （G247S, E280G, P362T and A434D） of phenylalanine hydroxylase gene （PAH）. Methods （ 1 ） The enzyme activity of the 4 mutants was assessed by using transient protein expression in mammalian ceils. （2） The PAH amino acid sequences among different animal species were alignmented. （3） The effects of the 4 missense mutations on the protein structure were analyzed. （4） The clinical phenotype of the patients with PKU were analyzed, according to their blood Phe levels prior to treatment and the Phe tolerance. Results （ 1 ） The residual enzyme activity expressed in vitro of G247S, E280G, P362T and A434D were 3.1%, 0.4%, 8.2% and 21.7% of the wildtype PAIl respectively; （2）Gly247, Glu280 and Pm362 were among the highly conserved amino acids, while Ala434 was only moderately conserved; （3） As revealed by 3D structural analysis, G247S and E280G, being located at the active center of the enzyme, interfered with the binding of PAIl to BH4 and fermnsion respectively, while P362T and A434D affected the formation and stability of the dimer and the tetramer of PAH; （4） As shown by clinical phenotypic analysis, classical PKU were observed in patients carrying G247S and E280G, moderate PKU were observed in patients carrying A434D, whereas both classical and moderate PKU were observed in patients carrying P362T. Condltsion （1） The E280G, G247S, P362T and A434D are all disease-cansing mutations, with those located at the center of the enzyme displaying the most marked pathogenic effect; （2）The results of the structural analysis of the 3D molecule are consistent with the activity assessment of the enzyme expressedin vitro ; （3） The consistency is observed between the genotype, the enzymatic activity expressed in vitro and the clinical phenotype.

关 键 词：苯丙氨酸羟化酶 突变基因 残余酶活性 三维分子结构 

分 类 号：R346[医药卫生—基础医学]