YEAST CARBAMOYL-PHOSPHATE-SYNTHETASE-ASPARTATE-TRANSCARBAMYLASE MULTIDOMAIN PROTEIN IS PHOSPHORYLATED INVITRO BY CAMP-DEPENDENT PROTEIN-KINASE
YEAST CARBAMOYL-PHOSPHATE-SYNTHETASE-ASPARTATE-TRANSCARBAMYLASE MULTIDOMAIN PROTEIN IS PHOSPHORYLATED INVITRO BY CAMP-DEPENDENT PROTEIN-KINASE
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DOI：
10.1111/j.1432-1033.1990.tb19376.x
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发表时间：
1990-10-24
期刊：
影响因子：
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通讯作者：
CARREY, EA
中科院分区：
文献类型：
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作者：
DENISDUPHIL, M;LECAER, JP;CARREY, EA
酿酒酵母中嘧啶从头合成的前两个步骤由 URA2 基因编码的多功能蛋白催化，该蛋白具有氨基甲酰磷酸 (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 转化细胞中纯化出的天然酶，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，发现单个 240 kDa 物质被磷酸化。该物种的胰蛋白酶消化在等电聚焦后产生单一的、酸性很强的磷酸肽。通过 HPLC 纯化，然后对该肽进行氨基酸测序，结果显示在预期共有序列 Arg-Arg-Phe-Ser 处有磷酸丝氨酸。对 URA2 基因序列的了解使得该位点位于二氢乳清酶样结构域和 ATCase 结构域之间的肽连接中。这样的位置可以解释为什么URA2蛋白的磷酸化既不改变CPSase和ATCase的活性，也不改变它们对UTP（它们的常见特异性抑制剂）的敏感性。
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.