Quantitative production of compound I from a cytochrome P450 enzyme at low temperatures. Kinetics, activation parameters, and kinetic isotope effects for oxidation of benzyl alcohol.
Quantitative production of compound I from a cytochrome P450 enzyme at low temperatures. Kinetics, activation parameters, and kinetic isotope effects for oxidation of benzyl alcohol.
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在低温下，来自细胞色素P450酶的化合物I定量产生。动力学，激活参数和动力学同位素效应氧化氧化。
DOI：
10.1021/ja9031105
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发表时间：
2009-08-05
影响因子：
15
通讯作者：
Newcomb, Martin
中科院分区：
文献类型：
--
作者：
Wang, Qin;Sheng, Xin;Horner, John H.;Newcomb, Martin
细胞色素P450酶通常被认为是通过一种称为化合物I的铁(IV)-氧卟啉自由基阳离子瞬态氧化底物，但P450化合物I的动力学研究基本上不存在。我们报道了在含有50%甘油的缓冲液中，细胞色素P450 119 （CYP119）在低温下由相应的化合物II物种通过365 nm脉冲灯光解生产化合物I。研究了化合物1在苯甲醇及其苯基单氘和双氘同位素体中的氧化作用。在- 50°C下，底物浓度在1.0到6.0 mM之间时获得的准一级速率常数显示出饱和动力学，该饱和动力学给出了化合物I的底物结合常数（Kbind）和氧化反应的一级速率常数（cox）。苯甲醇氧化的代表性结果为Kbind = 214 M−1和kox = 0.48 s−1。在- 50 ~ - 25℃范围内，研究了二氘化底物C6H5CD2OH的反应动力学，建立了络合反应的van't Hoff图和氧化反应的Arrhenius图。- 50°C时的H/D动力学同位素效应（KIEs）分为较大的初级KIE （P = 11.9）和较小的逆次级KIE （S = 0.96）。将22°C时C6H5CD2OH氧化速率常数和未氘化和已氘化底物的KIE值与先前在激光闪光光解实验中获得的值进行比较，表明隧道效应可能是−50°C时总速率常数的重要组成部分。
Cytochrome P450 enzymes are commonly thought to oxidize substrates via an iron(IV)-oxo porphyrin radical cation transient termed Compound I, but kinetic studies of P450 Compounds I are essentially non-existent. We report production of Compound I from cytochrome P450 119 (CYP119) in high conversion from the corresponding Compound II species at low temperatures in buffer mixtures containing 50% glycerol by photolysis with 365 nm light from a pulsed lamp. Compound I was studied as a reagent in oxidations of benzyl alcohol and its benzylic mono- and dideuterio isotopomers. Pseudo-first-order rate constants obtained at −50 °C with concentrations of substrates between 1.0 and 6.0 mM displayed saturation kinetics that gave binding constants for the substrate in the Compound I species (Kbind) and first-order rate constants for the oxidation reactions (kox). Representative results are Kbind = 214 M−1 and kox = 0.48 s−1 for oxidation of benzyl alcohol. For the dideuterated substrate C6H5CD2OH, kinetics were studied between −50 °C and −25 °C, and a van't Hoff plot for complexation and an Arrhenius plot for the oxidation reaction were constructed. The H/D kinetic isotope effects (KIEs) at −50 °C were resolved into a large primary KIE (P = 11.9) and a small, inverse secondary KIE (S = 0.96). Comparison of values extrapolated to 22 °C of both the rate constant for oxidation of C6H5CD2OH and the KIE for the non-deuterated and dideuterated substrates to values obtained previously in laser flash photolysis experiments suggested that tunneling could be a significant component of the total rate constant at −50 °C.
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影响因子：
3.6
作者：
Brandi, P;Galli, C;Gentili, P
通讯作者：
Gentili, P
影响因子：
3.7
作者：
Jung, C;Schünemann, V;Barra, AL
通讯作者：
Barra, AL
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15
作者：
Halgren, TA;Roberts, JD;Newcomb, M
通讯作者：
Newcomb, M
影响因子：
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作者：
Davydov, R;Macdonald, IDG;Hoffman, BM
通讯作者：
Hoffman, BM
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15
作者：
Ambundo, EA;Friesner, RA;Lippard, SJ
通讯作者：
Lippard, SJ