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

利用从番茄（Lycopersicon esculentum Mill.）中部分纯化的1-氨基环丙烷-1-羧酸（ACC）合成酶，研究了底物S-腺苷甲硫氨酸（SAM）对ACC合成酶的失活机理。水果，并获得了以下结果。(1)SAM分子有两种非对映体，（+）-和（-）-SAM。当比较它们作为ACC合酶的灭活剂的活性时，证明（+）-SAM比（-）-SAM快三倍地灭活该酶。(2)在一些产生乙烯的植物组织中检测到（+）-和（-）-SAM的含量。它们含有（-）-SAM，但不含（+）-SAM。因此，天然的（-）-SAM被认为是负责酶在体内的自动失活过程。(3)将酶与<14>在不同位置标记的13 C-SAM一起孵育，并分析掺入酶蛋白中的放射性。结果表明，SAM的2-氨基丁酸部分共价连接到ACC合酶。(4)在磷酸吡哆醛存在下，L-乙烯基甘氨酸显示出作为ACC合酶的不可逆灭活剂。这一发现与（3）中所述的发现相结合，表明SAM对ACC合酶的失活是通过形成作为中间体的乙烯基甘氨酸-ACC合酶复合物而进行的。提出了ACC合酶失活的机制。(5)用~（13）C-SAM特异性标记ACC合酶<14>，有利于分离ACC合酶活性部位放射性标记肽段，并进一步测定其氨基酸序列。

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.