Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel
Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel
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DOI：
10.1021/bi7023964
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发表时间：
2008-03-25
期刊：
影响因子：
2.9
通讯作者：
Peterson, Julian A.
中科院分区：
文献类型：
--
作者：
Haines, Donovan C.;Chen, Baozhi;Peterson, Julian A.
P450 BM-3是一种广泛研究的P450细胞色素，其天然融合到细胞色素P450还原酶结构域。该酶的血红素结构域的晶体结构先前已经产生了许多关于P450结构特征、底物结合特异性和底物结合时发生的构象变化的见解。虽然许多P450被咪唑抑制，但这种化合物不能有效地抑制P450 BM-3。以前已经发现ω-咪唑基脂肪酸是酶的弱抑制剂，并显示出与底物月桂酸的一些不寻常的协同性。我们开始通过将ω-咪唑基脂肪酸连接到氨基酸基团的氮上来改善这些抑制剂的性质，这是我们以前用来增加底物效力的策略。所得的抑制剂比其缺乏氨基酸基团的母体化合物显着更有效。一个新的抑制剂绑定到血红素结构域的P450 BM-3的晶体结构表明，与酶的氨基酸基团的相互作用的模式是不同于先前观察到的酰基氨基酸底物。此外，活性位点中的残基需要移动以容纳咪唑基团，这为咪唑本身的低亲和力提供了解释。最后，先前观察到的与月桂酸的协同性被解释为一个令人惊讶的开放的基板访问通道内衬疏水残基，可以潜在地容纳月桂酸除了抑制剂本身。
P450BM-3 is an extensively studied P450 cytochrome that is naturally fused to a cytochrome P450 reductase domain. Crystal structures of the heme domain of this enzyme have previously generated many insights into features of P450 structure, substrate binding specificity, and conformational changes that occur on substrate binding. Although many P450s are inhibited by imidazole, this compound does not effectively inhibit P450BM-3. omega-Imidazolyl fatty acids have previously been found to be weak inhibitors of the enzyme and show some unusual cooperativity with the substrate lauric acid. We set out to improve the properties of these inhibitors by attaching the omega-imidazolyl fatty acid to the nitrogen of an amino acid group, a tactic that we used previously to increase the potency of substrates. The resulting inhibitors were significantly more potent than their parent compounds lacking the amino acid group. A crystal structure of one of the new inhibitors bound to the heme domain of P450BM-3 reveals that the mode of interaction of the amino acid group with the enzyme is different from that previously observed for acyl amino acid substrates. Further, required movements of residues in the active site to accommodate the imidazole group provide an explanation for the low affinity of imidazole itself. Finally, the previously observed cooperativity with lauric acid is explained by a surprisingly open substrate-access channel lined with hydrophobic residues that could potentially accommodate lauric acid in addition to the inhibitor itself.