Action Mechanism of Suicide Inhibitors of Ascorbate Peroxidase

1.利用两种过氧化物酶对抑制剂和底物的不同特性，建立了分离测定两种过氧化物酶以及叶绿体和胞质中抗坏血酸过氧化物酶同工酶的简便方法.二硫苏糖醇、谷胱甘肽、巯基乙醇和半胱氨酸等巯基化合物对茶叶中APX的过氧化氢氧化有抑制作用。这些硫醇本身并不使酶变性。然而，当过氧化氢是由金属催化氧化硫醇或当外源性过氧化氢被添加时，他们失活的酶。巯基被APX和过氧化氢氧化成巯基自由基。巯基和过氧化氢使APX失活是由于酶与反应中心产生的巯基相互作用引起的.用赖氨酰内肽酶对茶树叶片叶绿体APX同工酶进行酶切，并对酶切片段的氨基酸序列进行了测定。APX多肽序列与植物细胞色素c过氧化物酶的同源性高于愈创木酚过氧化物酶.从菠菜叶中发现了类囊体结合形式的APX。结合形式的活性与在siroma中的活性为1：1。类囊体酶主要定位于基质类囊体中，并且仅被洗涤剂溶解。类囊体结合的APX的酶性质是非常相似的基质APX。

1. Using different characteristics as to inhibitors and substrates, simple methods for separate assays of the two types of peroxidases, and of chloroplast and cytosol isizymes of ascorbate peroxidases (APX) were developed.2. The hydrogen peroxide-dependent oxidation of APX from tea leaves was inhibited by thiols, such as dithiothreitol, glutathione, mercaptoethanol and cysteine. These thiols themselves did not inactivate the enzyme. However, they inactivated the enzyme when hydrogen peroxide was produced by the metal-catalyzed oxidation of thiols or when exogenous hydrogen peroxide was added. Thiols were oxidized by APX and hydrogen peroxide to thiol radicals. Inactivation of APX by thiols and hydrogen peroxide is caused by the interaction of the enzyme with the thiyl radicals produced at its reaction center.3. The thloroplast isozyme of APX from tea leaves was digested with lysyl endopeptidase, and the amino acid sequences of the peptide fragments were determined. The sequence of the peptides from APX exhibit a higher degree of homology to the sequences of cytochrome c peroxidase than to those of guaiaeol peroxidases from plants.4. A thylakoid-bound form of APX was found from spinach leaves. The activity of the bound form and that in siroma was 1 : 1. The thylakoid enzyme was predominantly localized in the stroma thylakoids, and solubilized only by detergents. Enzymatic properties of the thylakoid-bound APX were very similar to those of the stromal APX.