Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type.
Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type.
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酶催化五氯乙烷脱卤：为什么 F87W-细胞色素 P450cam 比野生型更快。
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
Rick L. Ornstein
中科院分区：
文献类型：
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作者：
John I. Manchester;Rick L. Ornstein
在厌氧条件下，细胞色素P450可以还原脱卤重卤代烃，如一碳和二碳有机溶剂。这种催化能力引起了人们对工程形式的P450在修复受污染的深层地下生态系统中的潜在用途的关注。Loida（1994，PhD Thesis，University of Illinois at Urbana-Champaign，IL）和S.G. Sligar（个人交流）最近观察到细胞色素P450 cam（F87 W）的活性位点变体使五氯乙烷脱氯的速度比野生型酶快大约三倍。分子动力学模拟表明，突变酶的结合口袋仍然较小，五氯乙烷假设配置更接近血红素-Fe的F87 W突变体的两倍，往往在野生型酶。该结果与脱卤的碰撞模型一致，该模型与实验观察结果[Li和Wackett（1993）Biochemistry，32，9355-9361]一致，即含有野生型P450的溶液可以比含有游离血红素的溶液快100倍地使五氯乙烷脱卤。模拟表明，在碳-卤素键还原过程中，Trp 87不太可能显著稳定底物上的负电荷。结合空间和电子效应的改进微生物酶的设计继续用于原位修复卤化污染物。
Under anaerobic conditions, cytochromes P450 can reductively dehalogenate heavily halogenated hydrocarbons, such as one- and two-carbon organic solvents. This catalytic capacity has drawn attention to the potential use of engineered forms of P450s in the remediation of contaminated deep subsurface ecosystems. Loida (1994, PhD Thesis, University of Illinois at Urbana-Champaign, IL) and S.G. Sligar (personal communication) have observed recently that an active-site variant of cytochrome P450cam (F87W) dechlorinates pentachloroethane approximately three times faster than the wild-type enzyme. Molecular dynamics simulations have revealed that the mutant enzyme binding pocket remains smaller, and that pentachloroethane assumes configurations closer to the heme-Fe in the F87W mutant twice as often as in the wild-type enzyme. This result is consistent with a collisional model of dehalogenation, which agrees with experimental observations [Li and Wackett (1993) Biochemistry, 32, 9355-9361] that solutions containing wild-type P450cam dehalogenate pentachloroethane 100 times faster than those containing free heme. The simulations suggest that it is unlikely that Trp87 significantly stabilizes the developing negative charge on the substrate during carbon-halogen bond reduction. The design of improved microbial enzymes that incorporate both steric and electronic effects continues for use in remediating halogenated contaminants in situ.