Rescue of the horseradish peroxidase His-170-->Ala mutant activity by imidazole: importance of proximal ligand tethering.
Rescue of the horseradish peroxidase His-170-->Ala mutant activity by imidazole: importance of proximal ligand tethering.
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通过咪唑拯救辣根过氧化物酶 His-170-->Ala 突变体活性：近端配体束缚的重要性。
DOI：
10.1021/bi9609331
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
OrtizdeMontellano,PR
中科院分区：
文献类型：
--
作者：
Newmyer,SL;Sun,J;Loehr,TM;OrtizdeMontellano,PR
辣根过氧化物酶（HRP）中的近端铁配体是His-170。多组氨酸标记的HRP（hHRP）的H170 A突变体已在杆状病毒系统中表达，并已纯化和表征。在pH 7时，突变体的Soret最大吸收峰在414 nm，而不是403 nm。共振拉曼光谱表明，该蛋白质主要是6-坐标低自旋的铁态与带在亚铁态在212 cm-1表示远端组氨酸配位的铁。外源咪唑（Im）与H170 A-hHRP的结合常数Kd = 22 ± 4 mM，H170 A-hHRP与H2 O2反应时，不能产生光谱上可检测到的化合物I或化合物II中间体，但会导致血红素基团的逐渐降解。然而，H170 A HRP是催化活性的，其愈创木酚和ABTS过氧化物酶活性分别提高260和125倍，在饱和浓度的Im的存在下。Im对愈创木酚和ABTS的刺激作用的Km均为24 mM。H170 A hHRP的pH曲线与野生型hHRP的pH曲线不同，但这些差异基本上被Im消除。用稳态动力学方法测定了H170 A hHRP的“化合物I”生成速率，无Im时k1 = 16 M-1 s-1，有Im时k1 = 2.4 × 104 M-1 s-1。野生型hHRP的相应速率为k_1 = 4.4 × 106 M ~（-1）s ~（-1）。结果表明，Im结合在由H170 A突变产生的空腔中，与血红素铁原子配位，并通过挽救化合物I形成的速率来恢复大部分催化活性。然而，这种救援的催化活性的Im可能是有限的血红素的远端组氨酸（His-42）的H170 A突变体的协调。因此，近端组氨酸的主要功能是束缚铁原子以不利于第六配体结合，特别是铁与远端组氨酸的配位。此外，在化合物I的形成中，邻近配体的强氢键可能对促进O−O键断裂至关重要。
The proximal iron ligand in horseradish peroxidase (HRP) is His-170. The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized. At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm. Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1indicative of distal histidine coordination to the iron. Exogenous imidazole (Im) binds to the enzyme withKd= 22 ± 4 mM. Reaction of H170A hHRP with H2O2does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group. Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im. TheKmfor the stimulatory effect of Im is 24 mM for both guaiacol and ABTS. The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im. The rate of formation of “compound I” for H170A hHRP, determined by steady state kinetic methods, isk1= 16 M-1s-1without Im andk1= 2.4 × 104M-1s-1with Im. The corresponding rate for wild-type hHRP isk1= 4.4 × 106M-1s-1. The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation. However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant. Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine. In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O−O bond cleavage in the formation of compound I.