Davydov Dmitri R, Kumar Santosh, Halpert James R
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77555-1031, USA.
Biochem Biophys Res Commun. 2002 Jun 21;294(4):806-12. doi: 10.1016/S0006-291X(02)00565-X.
1-Pyrenebutanol (1-PB) has been used as a new fluorescent substrate for P450eryF to explore the molecular mechanisms of cooperativity. Hydroxylation of 1-PB by P450eryF was detected by both fluorometric and chromatographic assays. Binding was monitored by a substrate-induced low-to-high spin shift, as well as by fluorescence resonance energy transfer (FRET) from 1-PB to the heme. Spectrophotometric titration showed that P450eryF has high affinity for 1-PB with distinct positive cooperativity (S(50) = 12.4 +/- 2.2 microM, n = 2.3 +/- 0.6), as also revealed in activity measurements. FRET analysis showed a different binding process obeying a simple bimolecular mechanism with a K(D) = 2.15 +/- 0.8 microM that suggests the presence of the higher affinity binding site. 1-PB binding at this site appears not to modulate the spin state directly but rather to facilitate the spin shift caused by the interactions of P450eryF with the other substrate molecule.
1-芘丁醇(1-PB)已被用作细胞色素P450eryF的新型荧光底物,以探究协同作用的分子机制。通过荧光法和色谱法检测P450eryF对1-PB的羟基化作用。通过底物诱导的低自旋到高自旋转变以及从1-PB到血红素的荧光共振能量转移(FRET)来监测结合情况。分光光度滴定表明,P450eryF对1-PB具有高亲和力,并具有明显的正协同性(S(50) = 12.4 ± 2.2 μM,n = 2.3 ± 0.6),这在活性测量中也有所体现。FRET分析显示了一个不同的结合过程,遵循简单的双分子机制,K(D) = 2.15 ± 0.8 μM,这表明存在更高亲和力的结合位点。1-PB在此位点的结合似乎不会直接调节自旋状态,而是促进由P450eryF与其他底物分子相互作用引起的自旋转变。