Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3.
Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3.
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DOI：
10.1021/bi980462d
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发表时间：
1998-11
期刊：
影响因子：
2.9
通讯作者：
M. Noble;L. Quaroni;G. Chumanov;K. Turner;S. Chapman;R. Hanzlik;A. Munro
中科院分区：
文献类型：
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作者：
M. Noble;L. Quaroni;G. Chumanov;K. Turner;S. Chapman;R. Hanzlik;A. Munro
ω-咪唑基羧酸（C10-C12）已被用作巨大芽孢杆菌脂肪酸羟化酶P-450 BM 3活性位点和催化机制的探针。这些化合物是迄今报道的P-450 BM 3的最有效的抑制剂。所有这些都是混合抑制剂，增加了Km，降低了月桂酸氧化的kcat。所有连接P-450 BM 3三价铁血红素铁，诱导Soret吸收带从419至424 nm的II型位移。与含铁形式的结合要弱得多。10-（咪唑基）癸酸是最好的抑制剂（Kic = 0.9 microM，Kiu = 5.7 microM），而12-（咪唑基）十二烷酸（Kic = 1.35 microM，Kiu = 6.9 microM）优于11-（咪唑基）十一烷酸（Kic = 7.5 microM，Kiu = 16 microM）的上级。结合到氧化的P-450 BM 3血红素铁的解离常数通过电化学计量法测定为8 μ M（C12唑）和27 μ M（C11唑）。10-（咪唑基）癸酸的结合太紧密，无法通过荧光光度法测定绝对Kd，但该值<0.2 μ M。不同的脂肪酸的酶的结合被发现有不同的影响上的Kd的唑。月桂酸酯诱导更紧密的结合（C12唑的Kd降低至4.7 μ M），而花生四烯酸酯减弱亲和力（Kd增加至23 μ M）。花生四烯酸盐充分降低了对C10唑的亲和力，因此可以通过分光光度滴定法测定Kd（11 μ M）。在活性位点突变体R47 G（R47系链脂肪酸羧酸基团）和F87 Y中，C12唑的亲和力降低，但在突变体F87 G中增加，这表明该残基在确定血红素可及性中的重要作用。C10唑与自旋状态不敏感的F87 Y的结合要弱得多（32. 2 μ M），这表明抑制剂可能优先结合P-450 BM 3的不同构象。还原酶中的NADP+结合也收紧了这些抑制剂对P-450 BM 3的亲和力（C12唑的Kd降低至2.7 μ M），但在FMN缺陷突变体W574 D中没有观察到这种效应，表明NADP+对抑制剂结合的结构域间效应是通过黄素单核苷酸肽介导的。共振拉曼光谱表明，抑制剂与P-450 BM 3形成低自旋复合物，并且它们的结合诱导血红素乙烯基相对于环的运动。
omega-Imidazolyl carboxylic acids (C10-C12) have been used as probes of the active site and catalytic mechanism of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium. These compounds are the most potent inhibitors of P-450 BM3 yet reported. All are mixed inhibitors, increasing the Km and decreasing the kcat for laurate oxidation. All ligate the P-450 BM3 ferric heme iron, inducing a type II shift in the Soret absorbance band from 419 to 424 nm. Binding to the ferrous form is much weaker. 10-(Imidazolyl)decanoic acid was the best inhibitor (Kic = 0.9 microM, Kiu = 5.7 microM), while 12-(imidazolyl)dodecanoic acid (Kic = 1.35 microM, Kiu = 6.9 microM) was superior to 11-(imidazolyl)undecanoic acid (Kic = 7.5 microM, Kiu = 16 microM). Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 microM (C12 azole) and 27 microM (C11 azole). The binding of 10-(imidazolyl)decanoic acid was too tight for an absolute Kd to be determined spectrophotometrically, but this value is <0.2 microM. The binding of different fatty acids to the enzyme was found to have distinct effects on the Kd for the azoles. Laurate induced tighter binding (Kd for the C12 azole lowered to 4.7 microM), while arachidonate weakened the affinity (Kd increased to 23 microM). Arachidonate diminished the affinity for the C10 azole sufficiently that a Kd could be determined by spectrophotometric titration (11 microM). Affinity for the C12 azole was decreased in active-site-mutants R47G (R47 tethers the fatty acid carboxylate group) and F87Y but increased in mutant F87G-indicating an important role for this residue in determining heme accessibility. The C10 azole binds much more weakly to the spin-state-insensitive F87Y (32. 2 microM), suggesting that the inhibitors may bind preferentially to different conformers of P-450 BM3. NADP+ binding in the reductase also tightened affinity of these inhibitors for P-450 BM3 (Kd for the C12 azole decreased to 2.7 microM), but this effect was not observed for FMN-deficient mutant W574D, suggesting that the interdomain effect of NADP+ on inhibitor binding was mediated via flavin mononucleotide. Resonance Raman spectroscopy indicates that the inhibitors form low-spin complexes with P-450 BM3 and that their binding induces movements of the heme vinyls relative to the ring.