Denis-Duphil M, Lecaer J P, Hardie D G, Carrey E A
Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Eur J Biochem. 1990 Oct 24;193(2):581-7. doi: 10.1111/j.1432-1033.1990.tb19376.x.
The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl-phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease-B-deficient URA2-transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP-dependent protein kinase. After electrophoresis under denaturing conditions, a single 240-kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg-Arg-Phe-Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase-like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor.
酿酒酵母中从头嘧啶合成的前两个步骤由一种多功能蛋白催化，该蛋白由URA2基因编码，具有氨甲酰磷酸（CPSase）合成酶和天冬氨酸转氨甲酰酶（ATCase）活性。从缺乏蛋白酶B的URA2转化细胞中纯化的天然酶，在体外使用纯cAMP依赖性蛋白激酶的催化亚基进行磷酸化。在变性条件下进行电泳后，发现单一的240 kDa条带被磷酸化。该条带经胰蛋白酶消化后，在等电聚焦时产生单一的、酸性很强的磷酸肽。通过HPLC纯化并对该肽进行氨基酸测序，结果显示在预期的共有序列Arg-Arg-Phe-Ser处有一个磷酸丝氨酸。URA2基因序列的信息使该位点定位在二氢乳清酸酶样结构域和ATCase结构域之间的肽链中。这样的定位可能解释了为什么URA2蛋白的磷酸化既没有改变CPSase和ATCase的活性，也没有改变它们对其共同特异性抑制剂UTP的敏感性。
