Roach M P, Matsui T, Watanabe Y
Institute for Molecular Science, and Department of Structural Molecular Science, The Graduate University for Advanced Studies, Myodaiji, Okazaki, 444-8585, Japan.
Acc Chem Res. 2001 Oct;34(10):818-25. doi: 10.1021/ar9502590.
To pursue structure-function relationships of heme enzymes in the activation of peroxides, we have chosen to use myoglobin as the framework for our molecular engineering studies. Comparison of the crystal structures of myoglobin and peroxidases reveals differences in the arrangement of amino acid residues in heme active sites. On the basis of these structural differences and the reaction mechanisms of peroxidases, we have converted myoglobin into a peroxidase-like enzyme by alternation of the heme distal pocket via site-directed mutagenesis. The replacement of the proximal histidine with cysteine and the exogenous substituted imidazoles slightly accelerates the peroxide O-O bond cleavage due to the electron donor characteristics. However, we have not observed an enhancement in the activation of peroxide by the proximal mutant with tyrosine, the exogenous phenolate, and benzoate. A clear understanding of the absolute role of the proximal ligand remains elusive.
为了探究血红素酶在过氧化物激活中的结构-功能关系，我们选择使用肌红蛋白作为分子工程研究的框架。肌红蛋白和过氧化物酶晶体结构的比较揭示了血红素活性位点氨基酸残基排列的差异。基于这些结构差异和过氧化物酶的反应机制，我们通过定点诱变改变血红素远端口袋，将肌红蛋白转化为类似过氧化物酶的酶。由于电子供体特性，用半胱氨酸取代近端组氨酸和外源性取代咪唑会稍微加速过氧化物O-O键的断裂。然而，我们没有观察到近端酪氨酸突变体、外源性酚盐和苯甲酸盐对过氧化物激活的增强作用。对近端配体的绝对作用仍缺乏清晰的认识。
