Cleavage Reaction Mechanis of Peroxydative Product in Biogeneration of Leaf Green Odour

当13-氢过氧亚油酸与茶树叶绿体共孵育时，可以检测到包括几何异构体在内的多种产物，尽管我们实验室对氢过氧化物裂解酶的裂解反应进行了广泛的探索，但该酶是否也进行异构化尚不清楚。用13-氢过氧亚油酸的醇衍生物13-氢过氧亚油醇（1）研究了这一机理。将合成的12-羟基-（3 z）-十二烯醛（2）与茶叶叶绿体孵育，发现12-羟基-（2 E）-十二烯醛（3）是2异构化反应的产物。这种异构化也被发现是由2与热灭活的叶绿体孵育引起的。此外，这种异构化活性可以用热乙醇提取。这些结果表明，异构化是由热稳定因子而不是酶引起的。该因子在碱性条件下被激活，但不受盐浓度的影响。当13-过氧化氢亚油醇（1）与茶叶绿体在抑制异构化的条件下孵育时，只有在裂解反应几乎停止后才形成3。在<18>此抑制条件下，将~（20）O-（1）与茶树叶绿体共孵育，<18>能有效地检测到~（20）O-（2）。这些结果表明，异构化反应是独立的由氢过氧化物裂解酶进行的裂解反应。根据这一发现，提出该酶在切割反应期间保留底物（1）的C-9位双键的顺式构型。事实上，当（9 E，11 E）-（1）与该酶孵育时，不发生反应，底物C-9位的双键必须被酶活性中心周围的配体识别，但对反应本身没有贡献。

When 13-hydroperoxylinoleic acid is incubated with tea chloroplast, several products including geometrical isomers can be detected.Although the cleavage reaction performed by hydroperoxide lyase was extensively explored in our laboratry, whether this enzyme also progress isomerization or not had notbeen known yet. Investigation of this mechanism was performed with an alcohol derivative of 13-hydroperoxylinoleic acid, 13-hydroperoxylinoleyl alcohol (1). Incubation of synthesized 12-hydroxy-(3z)-dodecenal (2) with tea chloroplast revealed that 12-hydroxy-(2E)-dodecenal (3) was the product of isomerization reaction of 2. This isomerization was found to be caused also by incubation of 2 with heat inactivated chloroplast. Furthermore, this isomerization activity could be extracted with heat ehanol. These results indicate that isomerization is occured by heat-stable factor(s) but not by enzyme(s). This factor(s) was activated in alkaline condition but not affected with salt concentration. When 13-hydroperoxylinoleyl alcohol (1) was incubated with tea chloroplast under conditions which suppresses isomerization, 3 was formed only after cleavage reaction was almostly ceased. And when ^<18>O-(1) was incubated with tea chloroplast under this suppressed condition ^<18>O-(2) was effectively detected. These results indicated that the isomerization reaction was independent from cleavage reaction performed by hydroperoxide lyase. From this finding it is proposed that this enzyme retain cis-configuration of double bond at C-9 position of substrate (1) during cleavage reaction. In fact, when (9E, 11E)-(1) was incubated with this enzyme, there occurred no reaction Double bond at C-9 position of substrate must be recognized by some ligand around active centers of the enzyme but have no contribution on the reaction itself.