Huang Liusheng, Wojciechowski Grzegorz, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.
J Biol Chem. 2006 Jul 14;281(28):18983-8. doi: 10.1074/jbc.M602307200. Epub 2006 May 1.
Oxidation of SCN-, Br-, and Cl- (X-) by horseradish peroxidase (HRP) and other plant and fungal peroxidases results in the addition of HOX to the heme vinyl group. This reaction is not observed with lactoperoxidase (LPO), in which the heme is covalently bound to the protein via two ester bonds between carboxylic side chains and heme methyl groups. To test the hypothesis that the heme of LPO and other mammalian peroxidases is protected from vinyl group modification by the hemeprotein covalent bonds, we prepared the F41E mutant of HRP in which the heme is attached to the protein via a covalent bond between Glu41 and the heme 3-methyl. We also examined the E375D mutant of LPO in which only one of the two normal covalent heme links is retained. The prosthetic heme groups of F41E HRP and E375D LPO are essentially not modified by the HOBr produced by these enzymes. The double E375D/D225E mutant of LPO that can form no covalent bonds is inactive and could not be examined. These results unambiguously demonstrate that a single heme-protein link is sufficient to protect the heme from vinyl group modification even in a protein (HRP) that is normally highly susceptible to this reaction. The results directly establish that one function of the covalent heme-protein bonds in mammalian peroxidases is to protect their prosthetic group from their highly reactive metabolic products.
辣根过氧化物酶（HRP）以及其他植物和真菌过氧化物酶可将硫氰酸根离子（SCN-）、溴离子（Br-）和氯离子（Cl-，统称X-）氧化，从而使次卤酸（HOX）添加到血红素乙烯基上。而在乳过氧化物酶（LPO）中未观察到这种反应，在LPO中，血红素通过羧基侧链与血红素甲基之间的两个酯键与蛋白质共价结合。为了验证LPO和其他哺乳动物过氧化物酶的血红素因血红素-蛋白质共价键而免受乙烯基修饰这一假说，我们制备了HRP的F41E突变体，其中血红素通过Glu41与血红素3-甲基之间的共价键与蛋白质相连。我们还研究了LPO的E375D突变体，其中仅保留了两个正常共价血红素连接中的一个。F41E HRP和E375D LPO的辅基血红素基团基本上不会被这些酶产生的HOBr修饰。无法形成共价键的LPO双突变体E375D/D225E没有活性，因此无法进行研究。这些结果明确表明，即使在通常对该反应高度敏感的蛋白质（HRP）中，单个血红素-蛋白质连接也足以保护血红素免受乙烯基修饰。结果直接证明，哺乳动物过氧化物酶中共价血红素-蛋白质键的一个功能是保护其辅基免受其高反应性代谢产物的影响。
