Lampe Jed N, Floor Stephen N, Gross John D, Nishida Clinton R, Jiang Yongying, Trnka Michael J, Ortiz de Montellano Paul R
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517, USA.
J Am Chem Soc. 2008 Dec 3;130(48):16168-9. doi: 10.1021/ja8071463.
Conformational dynamics are thought to play an important role in ligand binding and catalysis by cytochrome P450 enzymes, but few techniques exist to examine them in molecular detail. Using a unique isotopic labeling strategy, we have site specifically inserted a (13)C-labeled unnatural amino acid residue, (13)C-p-methoxyphenylalanine (MeOF), into two different locations in the substrate binding region of the thermophilic cytochrome P450 enzyme CYP119. Surprisingly, in both cases the resonance signal from the ligand-free protein is represented by a doublet in the (1)H,(13)C-HSQC spectrum. Upon binding of 4-phenylimidazole, the signals from the initial resonances are reduced in favor of a single new resonance, in the case of the F162MeOF mutant, or two new resonances, in the case of the F153MeOF mutant. This represents the first direct physical evidence for the ligand-dependent existence of multiple P450 conformers simultaneously in solution. This general approach may be used to further illuminate the role that conformational dynamics plays in the complex enzymatic phenomena exhibited by P450 enzymes.
构象动力学被认为在细胞色素P450酶的配体结合和催化过程中发挥重要作用，但用于详细研究其分子机制的技术却很少。我们采用独特的同位素标记策略，将一个（13）C标记的非天然氨基酸残基，即（13）C-对甲氧基苯丙氨酸（MeOF），位点特异性地插入嗜热细胞色素P450酶CYP119底物结合区域的两个不同位置。令人惊讶的是，在这两种情况下，无配体蛋白的共振信号在（1）H，（13）C-HSQC谱中均表现为双峰。在结合4-苯基咪唑后，对于F162MeOF突变体，初始共振信号减弱并有利于出现一个新的单一共振信号；对于F153MeOF突变体，则出现两个新的共振信号。这是溶液中同时存在多种依赖配体的P450构象异构体的首个直接物理证据。这种通用方法可用于进一步阐明构象动力学在P450酶所展现的复杂酶促现象中所起的作用。
