Barbosa P, Cialkowski M, O'Brien W E
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1991 Mar 15;266(8):5286-90.
Argininosuccinic aciduria is an inborn error of metabolism due to the genetic deficiency of argininosuccinate lyase. In order to determine the molecular basis for the disease, RNA isolated from cultured skin fibroblasts derived from four unrelated patients was reverse-transcribed and amplified using the polymerase chain reaction and the products were cloned and sequenced. Three single base missense mutations were identified: Arg111----Trp, Gln286----Arg, and Arg193----Gln. One single base amber mutation was identified at Gln454. One mutation involved a 13-base pair deletion within exon 13, and it was noted that the majority of the mature RNA derived from this allele was deleted for the entire exon rather than containing the exon with the 13 bases deleted. A final mutation was observed in which exon 2 was deleted from the mature RNA. The molecular basis for this deletion was not determined. Of the eight potential mutations present in the four cell lines studied, six mutations were identified and further data indicate that the remaining two unidentified mutations were different from those identified. Two site-directed mutations were created in the cDNA, Lys51----Asn and His89----Gln, and these were expressed in yeast. The Lys51 mutation caused an approximate 2-fold reduction in activity and the His89 mutation resulted in an approximate 10-fold reduction in activity. The combination of determination of naturally occurring mutations and the study of the effect of site-directed mutations on the activity of argininosuccinate lyase provide insight into the amino acid residues critical to the function of the enzyme.
精氨琥珀酸尿症是一种由于精氨琥珀酸裂解酶基因缺陷导致的先天性代谢紊乱。为了确定该疾病的分子基础，从四名无关患者培养的皮肤成纤维细胞中分离出RNA，通过聚合酶链反应进行逆转录和扩增，产物进行克隆和测序。鉴定出三个单碱基错义突变：Arg111→Trp、Gln286→Arg和Arg193→Gln。在Gln454处鉴定出一个单碱基琥珀突变。一个突变涉及外显子13内13个碱基对的缺失，并且注意到来自该等位基因的大多数成熟RNA整个外显子缺失，而不是包含缺失13个碱基的外显子。在成熟RNA中观察到最后一个突变，即外显子2缺失。该缺失的分子基础尚未确定。在所研究的四个细胞系中存在的八个潜在突变中，鉴定出六个突变，进一步的数据表明其余两个未鉴定的突变与已鉴定的突变不同。在cDNA中产生了两个定点突变，Lys51→Asn和His89→Gln，并在酵母中表达。Lys51突变导致活性降低约2倍，His89突变导致活性降低约10倍。对天然发生的突变的测定以及定点突变对精氨琥珀酸裂解酶活性影响的研究相结合，为了解对该酶功能至关重要的氨基酸残基提供了线索。
