Macdonald Isabel K, Badyal Sandip K, Ghamsari Lila, Moody Peter C E, Raven Emma Lloyd
Department of Chemistry, University of Leicester, UK.
Biochemistry. 2006 Jun 27;45(25):7808-17. doi: 10.1021/bi0606849.
Previous work [Sharp, K. H., et al. (2003) Nat. Struct. Biol. 10, 303-307] has revealed the location of the ascorbate binding site in ascorbate peroxidase and has identified hydrogen-bonding interactions to Arg172, Lys30, and the heme 6-propionate as important in formation of the enzyme-substrate complex. In this work, the individual and collective contributions of these hydrogen bond interactions have been dissected using site-directed mutagenesis, steady-state and pre-steady-state kinetics, X-ray crystallography, and modified substrate analogues. Steady-state and pre-steady-state kinetic data reveal that the hydrogen bonds to Arg172 and the heme 6-propionate play a major part in stabilization of the bound ascorbate but that the interaction with Lys30 plays only a minor role. Binding of aromatic substrates is not affected by substitutions at Arg172/Lys30. Neutralization or removal of electrostatic charge at (Lys30) or adjacent to (Lys31) the ascorbate site does not substantially disrupt the binding interaction. Substrate oxidation and reduction of Compounds I and II is still possible in the absence of Arg172, but at a much reduced level. Crystallographic data (to 1.8 A) for the R172A variant indicate that the molecular structure of the proposed [Sharp, K. H., et al. (2004) Biochemistry 43, 8644-8651] proton transfer pathway from the ascorbate to the heme is conserved, which accounts for the residual activity. The results are discussed in terms of our wider understanding of the structural features that control substrate binding specificity in other peroxidase enzymes.
先前的研究工作[夏普，K. H.等人（2003年），《自然结构生物学》10卷，303 - 307页]揭示了抗坏血酸过氧化物酶中抗坏血酸结合位点的位置，并确定了与精氨酸172、赖氨酸30以及血红素6 - 丙酸酯的氢键相互作用在酶 - 底物复合物形成中很重要。在本研究中，利用定点诱变、稳态和预稳态动力学、X射线晶体学以及修饰的底物类似物，剖析了这些氢键相互作用的个体和集体贡献。稳态和预稳态动力学数据表明，与精氨酸172和血红素6 - 丙酸酯的氢键在结合的抗坏血酸的稳定中起主要作用，但与赖氨酸30的相互作用仅起次要作用。芳香族底物的结合不受精氨酸172/赖氨酸30处取代的影响。在抗坏血酸位点（赖氨酸30）或其相邻位置（赖氨酸31）中和或去除静电荷并不会实质性破坏结合相互作用。在没有精氨酸172的情况下，底物氧化以及化合物I和II的还原仍然可能，但水平大大降低。R172A变体的晶体学数据（分辨率达1.8埃）表明，从抗坏血酸到血红素的质子转移途径的分子结构[夏普，K. H.等人（2004年），《生物化学》43卷，8644 - 8651页]是保守的，这解释了残余活性。根据我们对控制其他过氧化物酶底物结合特异性的结构特征的更广泛理解，对结果进行了讨论。
