Mandelman D, Jamal J, Poulos T L
Department of Molecular Biology and Biochemistry, University of California, Irvine 92697-3900, USA.
Biochemistry. 1998 Dec 15;37(50):17610-7. doi: 10.1021/bi981958y.
Chemical and mutagenic modification combined with X-ray crystallography has been used to probe the ascorbate binding site in ascorbate peroxidase (APX). Chemical modification of the single Cys residue in APX with Ellman's reagent (DTNB) blocks the ability of APX to oxidize ascorbate but not other small aromatic phenolic substrates. DTNB-modified APX (APX-TNB) exhibits only 1.3% wild-type activity when ascorbate is used as the substrate but full activity when aromatic substrates, guaiacol or pyrogallol, are used. Stopped-flow studies show that APX-TNB reacts normally with peroxide to give compound I but that the rates of reduction of both compounds I and II by ascorbate are dramatically slowed. Conversion of Cys32 to Ser leads to approximately 70% drop in ascorbate peroxidase activity with no effect on guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionic ascorbate molecule interact with different sites on APX. The 2.0 A X-ray crystal structure of APX-TNB shows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of the asymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blocked by DTNB modification which is well removed from the exposed delta-heme edge where aromatic substrates are thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does not occur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 and the heme propionates.
化学修饰和诱变修饰与X射线晶体学相结合，已被用于探究抗坏血酸过氧化物酶（APX）中的抗坏血酸结合位点。用埃尔曼试剂（DTNB）对APX中的单个半胱氨酸残基进行化学修饰，会阻断APX氧化抗坏血酸的能力，但不会影响其氧化其他小的芳香酚类底物的能力。当以抗坏血酸为底物时，DTNB修饰的APX（APX-TNB）仅表现出1.3%的野生型活性，但当使用芳香底物愈创木酚或邻苯三酚时则具有完全活性。停流研究表明，APX-TNB与过氧化物正常反应生成化合物I，但抗坏血酸对化合物I和II的还原速率显著减慢。将半胱氨酸32突变为丝氨酸会导致抗坏血酸过氧化物酶活性下降约70%，而对愈创木酚过氧化物酶活性没有影响。这些结果表明，不带电荷的芳香底物和阴离子抗坏血酸分子与APX上的不同位点相互作用。APX-TNB的2.0埃X射线晶体结构显示，在不对称单元的所有四个分子中，与半胱氨酸32共价连接的TNB基团都有清晰的电子密度，表明修饰完全且具有特异性。似乎抗坏血酸位点被DTNB修饰所阻断，该修饰远离芳香底物被认为结合的暴露的δ-血红素边缘。这是第一个实验证据，表明抗坏血酸氧化并非发生在暴露的血红素边缘，而是发生在靠近精氨酸172和血红素丙酸酯的半胱氨酸32附近的另一个结合位点。
