Enzymatic reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: Kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy
Enzymatic reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: Kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy
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DOI：
10.1021/bi011883p
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发表时间：
2002-02-12
期刊：
影响因子：
2.9
通讯作者：
Shiro, Y
中科院分区：
文献类型：
--
作者：
Matsunaga, I;Yamada, A;Shiro, Y
细胞色素P450(SPalpha)(CYP152B1)和P450(BSbeta)(CYP152A1)分别是从少神经鞘氨醇单胞菌和枯草芽孢杆菌中分离得到的，属于P450超家族，但催化羟基化反应，在该反应中，来自H_2O_2的氧原子被有效地引入到脂肪酸(如肉豆蔻酸)中。P450(SPalpha)产生100%的α-羟化(α-OH)产物，而P450(BSbeta)分别产生33%和67%的α-和β-羟化(β-OH)产物。以氢取代脂肪酸[2，2-d(2)-肉豆蔻酸和d(27)-肉豆蔻酸]为底物，研究了两株细菌P450的过氧化物酶反应。在P450(SPalpha)反应中，当使用[2,242]-肉豆蔻酸时，我们观察到分子间非竞争性动力学同位素效应对V-max(V-D=4.1)，这表明在催化循环中存在涉及脂肪酸a-氢的同位素敏感步骤。另一方面，(D)(V/K)被掩盖，这与通常的单加氧酶P450的特征形成鲜明对比。特征动力学特征可以用比底物解离更快的产物形成来解释。以d(27)-肉豆蔻酸为底物时，P450(BSbeta)反应也有相似的动力学同位素效应[V-D=4.9，(D)(Vik)类似于1]，表明两种过氧酶的反应机理是相同的。在肉豆蔻酸存在和不存在的情况下，铁和亚铁-CO形成的P450(BSbeta)的共振拉曼光谱数据表明，酶的催化口袋是极性的，从而允许底物的羧基靠近血红素的第六配体。在这些动力学同位素效应和光谱结果的基础上，我们讨论了过氧酶P450(SPalpha)和P450(BSbeta)催化脂肪酸α-和β-羟基化的可能机制。
Cytochromes P450(SPalpha) (CYP152B1) and P450(BSbeta) (CYP152A1), which are isolated from Sphingomonas paucimobilis and Bacillus subtilis, respectively, belong to the P450 superfamily, but catalyze hydroxylation reactions, in which an oxygen atom from H2O2 is efficiently introduced into fatty acids (e.g., myristic acid). P450(SPalpha) produces the alpha-hydroxylated (alpha-OH) products at 100%, while P450(BSbeta) produces alpha- and beta-hydroxylated (beta-OH) products at 33 and 67%, respectively. Using deuterium-substituted fatty acids ([2,2-d(2)]-myristic acid and d(27)-myristic acid) as a substrate, the peroxygenase reactions of the two bacterial P450s were investigated. In the P450(SPalpha) reaction, we observed an intermolecular noncompetitive kinetic isotope effect on V-max (V-D = 4.1) when [2,242]-myristic acid was used, suggesting that an isotopically sensitive step involving the a-hydrogen of the fatty acid is present in the catalytic cycle. On the other hand, (D)(V/K) was masked, in sharp contrast to the features of usual monooxygenases P450. The characteristic kinetic features can be interpreted in terms of the faster product formation than the substrate dissociation. A similar kinetic isotope effect was observed [V-D = 4.9, (D)(VIK) similar to 1] for the P450(BSbeta) reaction, when d(27)-myristic acid was used as a substrate, indicating that the reaction mechanism is the same for both peroxygenases. The resonance Raman spectral data of P450(BSbeta) in the ferric and ferrous-CO forms in the presence and absence of myristic acid demonstrated that the catalytic pocket of the enzyme is polar, so that the location of the carboxylate of the substrate close to the sixth ligand of the heme could be allowed. On the basis of these results on the kinetic isotope effects and spectroscopy, we discuss the possible mechanisms of the alpha- and beta-hydroxylation of fatty acids catalyzed by peroxygenases P450(SPalpha) and P450(BSbeta).