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Regulation of Ethylene Biosynthesis --- Mechanism for SAM-Induced Inactivation of ACC synthase

乙烯生物合成的调控——SAM诱导ACC合酶失活的机制

基本信息

批准号：
63540524
负责人：
SATOH Shigeru
金额：
$ 0.83万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

It was investigated the mechanism for the inactivation of 1-aminocyclopropane -1-carboxylate (ACC) synthase by the substrate, S-adenonosylmethionine (SAM), using an enzyme preparation partially purified from tomato ( Lycopersicon esculentum Mill.) fruits, and the following results were obtained.(1) SAM molecule has two sulfonium diastereomers, (+)- and (-)-SAM. When their activities as an inactivator of ACC synthase were compared, it was demonstrated that (+)-SAM inactivated the enzyme three times faster than (-)-SAM.(2) The contents of both (+)- and (-)-SAM were examined in some plant tissues, which produced ethylene at high rates. They contained (-)-SAM, but not (+)-SAM. Thus the natural (-)-SAM was thought to be responsible for the auto- inactivation process of the enzyme in vivo.(3) The enzyme was incubated with ^<14>C-SAM labeled at different positions, and the radioactivity incorporation into the enzyme protein was analyzed. It was revealed that 2-aminobutyric acid portion of SAM is linked covalently into ACC synthase.(4) L-Vinylglycine was shown to act as an irreversible inactivator of ACC synthase in the presence of pyridoxal phosphate. This finding coupled with that described in (3) suggested that the inactivation of ACC synthase by SAM proceeds through the formation of a vinylglycine-ACC synthase complex as an intermediate. A scheme for the mechanism-based inactivation of ACC synthase was postulated.(5) The specific labeling of ACC synthase by ^<14>C-SAM would facilitate the isolation of radio-labeled active-site peptide of ACC synthase and the subsequent determination of its amino-acid sequence.

采用从番茄（Lycopersicon esculentum Mill.）果实中部分纯化的酶制剂，研究了底物S-腺苷甲硫氨酸（SAM）灭活1-氨基环丙烷-1-羧酸（ACC）合酶的机制，得到了以下结果。（1）SAM分子具有两个锍非对映异构体， (+)-和(-)-SAM。当比较它们作为 ACC 合酶灭活剂的活性时，结果表明 (+)-SAM 灭活该酶的速度是 (-)-SAM 的三倍。(2) 在一些以高速率产生乙烯的植物组织中检测了 (+)- 和 (-)-SAM 的含量。它们含有 (-)-SAM，但不含 (+)-SAM。因此，天然的(-)-SAM被认为负责酶在体内的自动失活过程。(3)将酶与在不同位置标记的^ 14 C-SAM一起温育，并分析酶蛋白中的放射性掺入。结果表明，SAM 的 2-氨基丁酸部分与 ACC 合酶共价连接。(4) 在磷酸吡哆醛存在下，L-乙烯基甘氨酸可作为 ACC 合酶的不可逆灭活剂。这一发现与 (3) 中描述的结果相结合表明 SAM 对 ACC 合酶的灭活是通过形成乙烯基甘氨酸-ACC 合酶复合物作为中间体进行的。提出了基于机制的ACC合酶失活方案。(5)^ 14 C-SAM对ACC合酶的特异性标记将有助于ACC合酶放射性标记活性位点肽的分离以及随后其氨基酸序列的测定。