Relationship of active site topology to substrate specificity for cytochrome P450terp (CYP108).
Relationship of active site topology to substrate specificity for cytochrome P450terp (CYP108).
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DOI：
10.1016/s0021-9258(19)61979-4
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发表时间：
1994-11
期刊：
影响因子：
--
通讯作者：
J. Fruetel;R. Mackman;J. Peterson;P. Ortiz de Montellano
中科院分区：
文献类型：
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作者：
J. Fruetel;R. Mackman;J. Peterson;P. Ortiz de Montellano
早期的研究表明，细胞色素P450与芳基肼的反应产生芳基-铁络合物，并且芳基向血红素的吡咯氮的氧化迁移提供了活性位点拓扑结构的信息。用这种方法比较细胞色素P450 terp（CYP 108）、P450 cam（CYP 101）和P450 BM-3（CYP 102）表明，P450 terp的活性位点比其他两种酶的活性位点更有效地受到空间限制，并且主要在血红素基团的吡咯环D上方开放。P450 terp活性位点的实验模型与X射线晶体学推导的模型不同，这表明吡咯环C也相对开放。结果表明，芳基位移可以用来探测构象的活性位点以外的被困在晶体状态。通过P450 terp从α-萜品醇形成的产物的鉴定表明，该酶专门羟基化最空间可及的、烯丙基活化的位置。该酶还将与α-萜品醇无关的取代的苯硫醚和苯乙烯氧化成相应的亚砜和环氧化物。在4-甲硫基苯甲醚和4-甲基苯乙烯的情况下，甲基羟基化在P450 terp催化的反应中有效地与磺基氧化和环氧化竞争，而不是P450 BM-3或P450 cam催化的那些。通过比较P450 terp、P450 cam和P450 BM-3对苯硫醚磺基氧化和苯乙烯环氧化的立体选择性，发现P450 terp的立体选择性最高，P450 BM-3的立体选择性最低。通过P450 terp进行的茴香硫醚磺基氧化的立体特异性取决于对位取代基的电子性质，并且从p-MeO的20：80的（R）：（S）比上升到p-CN的< 01：99的值。对于所研究的两个取代基，由P450 terp产生的环氧化物的（R）：（S）比约为90：10。细胞色素P450 cam和P450 BM-3的立体选择性要小得多。一个模型建议的立体化学和拓扑数据的结合底物P450 terp。
Earlier studies have shown that the reactions of cytochrome P450 with arylhydrazines yield aryl-iron complexes, and that oxidative migration of the aryl groups to the pyrrole nitrogens of the heme provides information on the active site topology. Comparison of cytochromes P450terp (CYP108), P450cam (CYP101), and P450BM-3 (CYP102) by this method suggests that the active site of P450terp is effectively more sterically restricted than those of the other two enzymes and is primarily open above pyrrole ring D of the heme group. This experimental model of the P450terp active site differs from that deduced by x-ray crystallography, which shows that pyrrole ring C is also relatively open. The results suggest that aryl shifts can be used to probe conformations of the active site other than that trapped in the crystal state. Identification of the product formed from alpha-terpineol by P450terp shows that the enzyme exclusively hydroxylates the most sterically accessible, allylically activated position. The enzyme also oxidizes substituted thioanisoles and styrenes unrelated to alpha-terpineol to the corresponding sulfoxides and epoxides. In the case of 4-methylthioanisole and 4-methylstyrene, methyl hydroxylation competes effectively with sulfoxidation and epoxidation in the reaction catalyzed by P450terp but not those catalyzed by P450BM-3 or P450cam. Comparison of the stereoselectively of thioanisole sulfoxidation and styrene epoxidation by P450terp, P450cam, and P450BM-3 shows that P450terp is the most, and P450BM-3 the least, stereospecific. The stereospecificity of thioanisole sulfoxidation by P450terp depends on the electronic nature of the para-substituent and rises from an (R):(S) ratio of 20:80 for p-MeO to a value of < 01:99 for p-CN. The (R):(S) ratio for the epoxides produced by P450terp is approximately 90:10 for the two substituents investigated. Cytochromes P450cam and P450BM-3 are much less stereoselective. A model is suggested by the stereochemical and topological data for the binding of substrates in P450terp.