The inactivation and catalytic pathways of horseradish peroxidase with m-chloroperoxybenzoic acid - A spectrophotometric and transient kinetic study
The inactivation and catalytic pathways of horseradish peroxidase with m-chloroperoxybenzoic acid - A spectrophotometric and transient kinetic study
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DOI：
10.1074/jbc.272.9.5469
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发表时间：
1997-02-28
影响因子：
4.8
通讯作者：
Arnao, MB
中科院分区：
文献类型：
--
作者：
RodriguezLopez, JN;HernandezRuiz, J;Arnao, MB
用常规和停流分光光度法研究了辣根过氧化物酶C(HRP-C)与间氯过氧苯甲酸(MCPBA)反应的催化循环和不可逆失活动力学。在pH 7.0，25℃时，间氯过氧苯甲酸将HRP-C氧化成化合物I，二级速率常数k(1)=3.6×10(7)M(-1)S(-1)，过量的间氯过氧苯甲酸作为单电子还原底物，将化合物I转化为化合物II，化合物II转化为静息铁酶。在这两个反应中，观察到了光谱上不同的瞬时形式的酶(化合物I与mCPB-4的λ(Max)=411 nm，epsilon=45 mm(-1)cm(-1)，以及化合物II的lambda(Max)=408 nm，epsilon=77 mm(-1)cm(-1))，化合物I-mCPBA中间体(近红外光谱显示与P965相同)在催化循环(k(3)=6.4×10(-3)S(-1))中衰变为化合物II，马鞭铁蛋白(k(I)=3.3x10(-3)S(-1))。因此，mCPBA对HRP-C铁的失活分配比为r=2，化合物II与mCPBA形成的中间体不是失活途径的一部分，只是通过催化循环衰变得到静止的铁酶(k(5)=1.0×10(-3)S(-1))。这些数据与以前的稳态动力学研究的数据进行了比较，证明了单一周转实验的重要性，并根据植物过氧化物酶与过氧化氢的生理相关反应对结果进行了讨论。
The kinetics of the catalytic cycle and irreversible inactivation of horseradish peroxidase C (HRP-C) reacting with m-chloroperoxybenzoic acid (mCPBA) have been studied by conventional and stopped-flow spectrophotometry, mCPBA oxidized HRP-C to compound I with a second order-rate constant k(1) = 3.6 x 10(7) M(-1) s(-1) at pH 7.0, 25 degrees C. Excess mCPBA subsequently acted as a one-electron reducing substrate, converting compound I to compound II and compound II to resting, ferric enzyme. In both of these reactions, spectrally distinct, transient forms of the enzyme were observed (lambda(max) = 411 nm, epsilon = 45 mM(-1) cm(-1) for compound I with mCPB-4, and lambda(max) = 408 nm, epsilon = 77 mM(-1) cm(-1) for compound II with mCPBA), The compound I-mCPBA intermediate (shown by near infrared spectroscopy to be identical to P965) decayed either to compound II in a catalytic cycle (k(3) = 6.4 x 10(-3) s(-1)) or, in a competing inactivation reaction, to verdohemoprotein (k(i) = 3.3 x 10(-3) s(-1)). Thus, a partition ratio of r = 2 is obtained for the inactivation of ferric HRP-C by mCPBA, The intermediate formed from compound II with mCPBA is not part of the inactivation pathway and only decays via the catalytic cycle to give resting, ferric enzyme (k(5) = 1.0 x 10(-3) s(-1)). The data are compared with those from earlier steady-state kinetic studies and demonstrate the importance of single turnover experiments, The results are discussed in terms of the physiologically relevant reactions of plant peroxidases with hydrogen peroxide.