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

Dmitri R. Davydov, Santosh Kumar, James R. Halpert

Research output: Contribution to journalArticle

30 Citations (Scopus)

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 (S50 = 12.4 ± 2.2 μM, 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 KD = 2.15 ± 0.8 μM 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.

Original languageEnglish (US)
Pages (from-to)806-812
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume294
Issue number4
DOIs
StatePublished - Jul 8 2002

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Substrates
Fluorescence Resonance Energy Transfer
Binding Sites
Hydroxylation
Heme
Titration
Assays
1-pyrenebutanol
Saccharopolyspora erythraea eryF protein
Molecules

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Allosteric mechanisms in P450eryF probed with 1-pyrenebutanol, a novel fluorescent substrate. / Davydov, Dmitri R.; Kumar, Santosh; Halpert, James R.

In: Biochemical and Biophysical Research Communications, Vol. 294, No. 4, 08.07.2002, p. 806-812.

Research output: Contribution to journalArticle

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