Improvement of peroxygenase activity by relocation of a catalytic histidine within the active site of horseradish peroxidase.
Improvement of peroxygenase activity by relocation of a catalytic histidine within the active site of horseradish peroxidase.
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DOI：
10.1021/bi9725780
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发表时间：
1998-07
期刊：
影响因子：
2.9
通讯作者：
M. Savenkova;J. Kuo;P. Ortiz de Montellano
中科院分区：
文献类型：
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作者：
M. Savenkova;J. Kuo;P. Ortiz de Montellano
为了研究Arg 38在辣根过氧化物酶（HRP）的过氧化和过氧化活性中的作用，我们表达了R38 A，R38 H和R38 H/H42 V突变体。R38 A HRP突变体产生正常的化合物I与H2 O2，其被亚铁氰化物还原为三价铁状态，而没有可检测的化合物II物质的形成。在R38 H和R38 H/H42 V突变体的情况下，化合物I本身仅通过停流法检测。对于R38 A、R38 H和R38 H/H42 V突变体，在4 ℃下化合物I形成的速率分别为8.0 × 10（4）、1.3 × 10（6）和1.6 × 10（3）M-1 s-1。R38 A、R38 H和R38 H/H42 V突变体氧化愈创木酚的速度分别比野生型酶慢10倍、2倍和55倍，氧化ABTS的速度比野生型酶慢6倍、3倍和32倍。茴香硫醚磺基氧化和苯乙烯环氧化的表观kcat/K（m）值表明R38 H和野生型酶的反应效率相当。然而，R38 A和R38 H/H42 V突变体作为磺基氧化催化剂的效率分别是野生型酶的190倍和1400倍，作为苯乙烯环氧化催化剂的效率分别是野生型酶的25倍和26倍。因此，尽管Arg 38在化合物I和II的形成和稳定中起作用，但其被其它残基取代可用于改善过氧化催化。
To examine the role of Arg38 in the peroxidative and peroxygenative activity of horseradish peroxidase (HRP), we have expressed the R38A, R38H, and R38H/H42V mutants. The R38A HRP mutant gives a normal compound I species with H2O2 that is reduced by ferrocyanide to the ferric state without the detectable formation of a compound II species. In the case of the R38H and R38H/H42V mutants, compound I itself is only detected by stopped flow methods. The rates of compound I formation at 4 degrees C are 8.0 x 10(4), 1.3 x 10(6), and 1.6 x 10(3) M-1 s-1 for the R38A, R38H, and R38H/H42V mutants, respectively. The R38A, R38H, and R38H/H42V mutants oxidize guaiacol 10-, 2-, and 55-fold, respectively, more slowly than the wild-type enzyme and oxidize ABTS 6-, 3-, and 32-fold more slowly than the wild-type enzyme. The apparent kcat/K(m) values for thioanisole sulfoxidation and styrene epoxidation indicate that the reaction efficiencies of the R38H and wild-type enzymes are comparable. However, the R38A and R38H/H42V mutants are 190- and 1400-fold more efficient as sulfoxidation catalysts, and 25- and 26-fold more efficient as styrene epoxidation catalysts, respectively, than the wild-type enzyme. Thus, even though Arg38 plays a role in the formation and stabilization of compounds I and II, its replacement by other residues can be used to improve peroxygenative catalysis.