Substitution of an amino acid residue axially coordinating to the heme molecule in hexameric tyrosine-coordinated hemoprotein to enhance peroxidase activity
Substitution of an amino acid residue axially coordinating to the heme molecule in hexameric tyrosine-coordinated hemoprotein to enhance peroxidase activity
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DOI：
10.1142/s1088424617500936
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发表时间：
2017-12-01
影响因子：
1.5
通讯作者：
Hayashi, Takashi
中科院分区：
文献类型：
--
作者：
Mashima, Tsuyoshi;Oohora, Koji;Hayashi, Takashi
为了在六聚体酪氨酸配位血红素蛋白中将原始酪氨酸配位血红素转化为组氨酸配位血红素，在蛋白质的每个亚基中分别用Phe45和His25替换HTHP、Tyr45（与血红素辅因子配位的残基）和位于远端位点的Arg25。通过SDS-PAGE、ESI-TOF MS、动态光散射和分子排阻色谱对突变株HTHP 25 H/Y45 F进行了表征。这些分析表明，HTHPR 25 H/Y45 F保持其稳定的六聚体结构与改变连接的每个血红素辅因子。HTHPR 25 H/Y45 F的铁、亚铁、CO和CN形式的紫外-可见吸收光谱与几种公知的His连接血红素蛋白的紫外-可见吸收光谱的比较表明，血红素由His 25残基配位。HTHPR 25 H/Y45 F与氢过氧化枯烯的反应以2.3：1的比例产生枯醇和苯乙酮，表明异裂O-O键断裂主要发生以形成称为化合物I的双电子氧化物质。发现HTHPR 25 H/Y45 F的过氧化物酶活性遵循Michaelis-Menten动力学。HTHPR 25 H/Y45 F对ABTS和愈创木酚的H2O2依赖性氧化的kcat值分别比野生型HTHP（HTHPWT）的kcat值高10倍和100倍。对于两种底物，HTHPR 25 H/Y45 F的k（cat）/K-m值相对于HTHPWT的k（cat）/K-m值增加30倍。HTHPR 25 H/Y45 F对茴香硫醚的氧化具有催化活性，其转化数约为100。2-比HTHPWT高一倍。目前的研究结果表明，近端His与血红素的连接对于增加HTHP基质中的过氧化物酶活性显着有效。
To convert an originally tyrosine-coordinated heme to histidine-coordinated heme in hexameric tyrosine-coordinated hemoprotein, HTHP, Tyr45, a residue coordinating to the heme cofactor, and Arg25 located in the distal site are replaced with Phe45 and His25, respectively in each of the subunits of the protein. The obtained HTHP mutant (HTHPR25H/Y45F) was characterized by SDS-PAGE, ESI-TOF MS, dynamic light scattering measurements and size exclusion chromatography. These analyses indicate that HTHPR25H/Y45F maintains its stable hexameric structure with the altered ligation of each of the heme cofactors. Comparison of UV-vis absorption spectra of the ferric-, ferrous-, CO- and CN-forms of HTHPR25H/Y45F with those of several well-known His-ligated hemoproteins indicates that heme is coordinated by the His25 residue. The reaction of HTHPR25H/Y45F with cumene hydroperoxide produces both cumyl alcohol and acetophenone in a 2.3: 1 ratio, indicating that heterolytic O-O bond cleavage dominantly occurs to form the two-electron oxidized species known as compound I. Peroxidase activity of HTHPR25H/Y45F is found to follow Michaelis-Menten kinetics. The kcat values of HTHPR25H/Y45F for H2O2-dependent oxidation of ABTS and guaiacol are 10-and 100-fold higher, respectively, than those of wild type HTHP (HTHPWT). The k(cat)/K-m values of HTHPR25H/Y45F for both substrates are increased 30-fold relative to that of HTHPWT. Moreover, HTHPR25H/Y45F is capable of promoting catalytic sulfoxidation of thioanisole with H2O2 with a turnover number ca. 2-fold higher than that of HTHPWT. The present findings demonstrate that proximal His ligation to the heme is significantly effective to increase the peroxidase activity in the HTHP matrix.