Adak S, Mazumder A, Banerjee R K
Department of Physiology, Indian Institute of Chemical Biology, Calcutta.
Biochem J. 1996 Mar 15;314 ( Pt 3)(Pt 3):985-91. doi: 10.1042/bj3140985.
The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate contents of 24M(-1.)min(-1), 0.8M(-1.)min(-1) and 0.54M(-1.)min(-1) respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0M(-1.)min(-1). The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement or arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (deltaG')for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete activation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35mM compared with 10mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichromism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.
通过化学修饰并对修饰后的酶进行表征，探究了辣根过氧化物酶（HRP）活性位点处精氨酸和酪氨酸残基在芳香族供体（愈创木酚）氧化中的可能作用。精氨酸特异性试剂苯乙二醛（PGO）、2,3 - 丁二酮和1,2 - 环己二酮均使该酶失活，遵循假一级动力学，二级速率常数分别为24M⁻¹·min⁻¹、0.8M⁻¹·min⁻¹和0.54M⁻¹·min⁻¹。用酪氨酸特异性试剂四硝基甲烷进行修饰，同样导致酶活性在假一级动力学下损失50%，二级速率常数为2.0M⁻¹·min⁻¹。底物H₂O₂以及电子供体如I⁻和SCN⁻对两种类型的修饰剂所致的失活均无保护作用，而该酶可被愈创木酚或邻联茴香胺完全保护，邻联茴香胺是被该酶氧化的一种芳香族电子供体（第二底物）。这些研究表明HRP芳香族供体位点处精氨酸和酪氨酸残基参与其中。愈创木酚保护的苯乙二醛修饰酶显示出与天然酶几乎相同的结合参数（Kd），以及供体结合时相似的自由能变化（ΔG'）。用[7 - ¹⁴C]苯乙二醛进行的化学计量学研究表明，每摩尔酶掺入2摩尔苯乙二醛，表明修饰一个精氨酸残基可实现完全失活。四硝基甲烷修饰酶与天然酶的差示吸收光谱在428nm处有一个峰，这是硝基酪氨酸残基的特征峰，用连二亚硫酸钠处理后该峰消失，表明酪氨酸残基发生了特异性修饰。失活化学计量学表明，每个酶分子修饰一个酪氨酸残基导致50%的失活。通过光学差示光谱进行的结合研究表明，精氨酸修饰的酶根本不能结合愈创木酚，而酪氨酸修饰的酶以降低的亲和力结合愈创木酚（Kd为35mM，而天然酶为10mM）。然而，两种修饰酶都保留了与H₂O₂形成化合物II（比天然高铁过氧化物酶高一价的单电子氧化态）的特性，但由于缺乏结合，在PGO修饰的酶中，愈创木酚不能将化合物II还原为天然酶。圆二色性研究未发现修饰导致蛋白质结构有非特异性变化。因此，我们认为HRP用于芳香族供体氧化的活性位点由一个精氨酸和一个相邻的酪氨酸残基组成，其中前者在芳香族供体结合中起关键作用，而后者残基可能通过疏水相互作用或氢键起促进作用。
