Suppr超能文献

用新型荧光底物1-芘丁醇探究P450eryF中的变构机制。

Allosteric mechanisms in P450eryF probed with 1-pyrenebutanol, a novel fluorescent substrate.

作者信息

Davydov Dmitri R, Kumar Santosh, Halpert James R

机构信息

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77555-1031, USA.

出版信息

Biochem Biophys Res Commun. 2002 Jun 21;294(4):806-12. doi: 10.1016/S0006-291X(02)00565-X.

Abstract

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.

摘要

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。

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验