Action Mechanism of Suicide Inhibitors of Ascorbate Peroxidase

1. 利用抗坏血酸过氧化物酶（APX）抑制剂和底物的不同特性，建立了两种过氧化物酶以及抗坏血酸过氧化物酶（APX）叶绿体和细胞质同工酶的分离测定方法。二硫代苏糖醇、谷胱甘肽、巯基乙醇和半胱氨酸抑制了茶叶中APX的过氧化氢依赖性氧化。这些硫醇本身并没有使酶失活。然而，当金属催化硫醇氧化产生过氧化氢或添加外源过氧化氢时，它们会使酶失活。硫醇被APX和过氧化氢氧化为硫醇自由基。硫醇和过氧化氢使APX失活是由酶与在其反应中心产生的硫基自由基相互作用引起的。用赖氨酸内肽酶对茶叶中APX的叶绿体同工酶进行酶切，并测定了肽片段的氨基酸序列。APX多肽序列与细胞色素c过氧化物酶序列的同源性高于与植物愈创木酚过氧化物酶序列的同源性。从菠菜叶中发现了类囊体结合形式的APX。结合形式的活度与siroma的活度为1:1。类囊体酶主要定位于基质类囊体，只能被洗涤剂溶解。类囊体结合的APX酶学性质与基质APX非常相似。

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.