Structural roles of the highly conserved glu residue in the heme distal site of peroxidases.
Structural roles of the highly conserved glu residue in the heme distal site of peroxidases.
复制标题
过氧化物酶血红素远端位点高度保守的谷氨酸残基的结构作用。
DOI：
10.1021/bi9725273
复制
发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
I. Morishima
中科院分区：
文献类型：
--
作者：
Motomasa Tanaka;K. Ishimori;I. Morishima
辣根过氧化物酶中的谷氨酸(Glu64)是多种真菌和植物过氧化物酶中血红素远端高度保守的氨基酸残基之一，它通过与水分子的氢键与远端钙离子相互作用，其主链上的多肽羰基氧通过相邻的天冬氨酸残基与远端组氨酸形成氢键网络，表明谷氨酸残基与钙离子的稳定和过氧化物酶的催化活性有关[Nagano，S.，Tanaka，M.，Ishimori，K.，Watanabe，Y.，and Morishima，I.(1996年)生化，35,14251-14258]。为了干扰与相邻天冬氨酸的氢键，我们用Pro(E64P)或Gly(E64G)取代了Glu，这会改变主链在位置的构型。这两个突变体在催化循环中对对苯二酚的氧化活性和基本反应速率都表现出显著的抑制作用。然而，E64S(Glu64->；Ser)突变体的主链构型和与Asn70的氢键不会受到影响，但与钙离子的相互作用因羧酸盐的去除而严重扰乱，与E64P和E64G突变体的催化活性也很低。E64S突变体的光谱特征与其他突变体相似：远端His的重定向，远端His与Asn70之间的氢键中断，以及钙离子的丢失。因此，我们可以得出结论，除了在远端形成氢键网络外，Glu残基是稳定结合钙离子的关键残基，它维持了远端空洞的结构完整性，导致了高的过氧化物酶活性。
One of the highly conserved amino acid residues in the heme distal site of various fungal and plant peroxidases, glutamic acid 64 (Glu64) in horseradish peroxidase (HRP), interacts with a distal calcium ion through a hydrogen bond with a water molecule and its peptide carbonyl oxygen on the main-chain forms the hydrogen bond network to the distal His via the adjacent Asn residue, suggesting that the Glu residue is related to the stabilization of the calcium ion and catalytic activity of peroxidase [Nagano, S., Tanaka, M., Ishimori, K., Watanabe, Y., and Morishima, I. (1996) Biochemistry 35, 14251-14258]. To perturb the hydrogen bond with the adjacent Asn, we replaced the Glu with Pro (E64P) or Gly (E64G), which would alter the configuration of the main chain at position 64. Both of the mutants exhibited substantially depressed oxidation activities for hydroquinone and elementary reaction rates in the catalytic cycle. However, the E64S (Glu64 --> Ser) mutant, in which the configuration of the main chain and the hydrogen bond with Asn70 would not be affected but the interactions with the calcium ion are seriously perturbed by removal of the carboxylate, also showed quite low catalytic activity as observed for the E64P and E64G mutants. Spectral features for the E64S mutant are similar to those of the other mutants: the reorientation of the distal His, disruption of the hydrogen bond between the distal His and Asn70, and loss of the calcium ion. Thus, we can conclude that, in addition to forming the hydrogen bond network in the distal site, the Glu residue is a key residue for stable binding of the calcium ion, which maintains the structural integrity of the distal cavity, resulting in high peroxidase activity.