Yang Hui-Jun, Matsui Toshitaka, Ozaki Shin-ichi, Kato Shigeru, Ueno Takafumi, Phillips George N, Fukuzumi Shunichi, Watanabe Yoshihito
Department of Structural Molecule Science, The Graduate University for Advanced Studies, Okazaki, Myodaiji 444-8585, Japan.
Biochemistry. 2003 Sep 2;42(34):10174-81. doi: 10.1021/bi034605u.
In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, we found that the replacement of His-64 of myoglobin (Mb) with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively. Since residue 68 is known to influence the ligation of small molecules to the heme iron, we constructed H64D/V68X Mb bearing Ala, Ser, Leu, Ile, and Phe at position 68 to improve the oxidation activity. The Val-68 to Leu mutation of H64D Mb accelerates the reaction with H(2)O(2) to form a catalytic species, called compound I, and improves the one-electron oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (i.e., peroxidase activity) approximately 2-fold. On the other hand, H64D/V68I Mb oxygenates thioanisole 2.7- and 1600-fold faster than H64D and wild-type Mb, respectively. In terms of the enantioselectivity, H64D/V68A and H64D/V68S Mb were good chiral catalysts for thioanisole oxidation and produced the (R)-sulfoxide dominantly with 84% and 88% ee, respectively [Kato, S., et al. (2002) J. Am. Chem. Soc. 124, 8506-8507]. On the contrary, the substitution of Val-68 in H64D Mb with an isoleucine residue alters the dominant sulfoxide product from the (R)- to the (S)-isomer. The crystal structures of H64D/V68A and H64D/V68S Mb elucidated in this study do not clearly indicate residues interacting with thioanisole. However, comparison of the active site structures provides the basis to interpret the changes in oxidation activity: (1) direct steric interactions between residue 68 and substrates (i.e., H(2)O(2), ABTS, thioanisole) and (2) the polar interactions between tightly hydrogen-bonded water molecules and substrates.
在阐明血红素附近区域对过氧化氢活化的结构要求时，我们发现用带负电荷的天冬氨酸残基取代肌红蛋白（Mb）的His-64，可分别使过氧化物酶和加氧酶活性提高78倍和580倍。由于已知68位残基会影响小分子与血红素铁的配位，我们构建了在68位带有丙氨酸、丝氨酸、亮氨酸、异亮氨酸和苯丙氨酸的H64D/V68X Mb，以提高氧化活性。H64D Mb的Val-68突变为Leu会加速与H₂O₂的反应，形成一种称为化合物I的催化物种，并使2,2'-偶氮双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）的单电子氧化（即过氧化物酶活性）提高约2倍。另一方面，H64D/V68I Mb使硫代苯甲醚的氧化速度分别比H64D和野生型Mb快2.7倍和1600倍。在对映选择性方面，H64D/V68A和H64D/V68S Mb是硫代苯甲醚氧化的良好手性催化剂，分别以84%和88%的对映体过量（ee）主要生成（R）-亚砜[加藤，S.等人（2002年）《美国化学会志》124，8506 - 8507]。相反，将H64D Mb中的Val-68用异亮氨酸残基取代会使主要的亚砜产物从（R）-异构体变为（S）-异构体。本研究阐明的H64D/V68A和H64D/V68S Mb的晶体结构并未明确表明与硫代苯甲醚相互作用的残基。然而，活性位点结构的比较为解释氧化活性的变化提供了依据：（1）68位残基与底物（即H₂O₂、ABTS、硫代苯甲醚）之间的直接空间相互作用，以及（2）紧密氢键结合的水分子与底物之间的极性相互作用。
