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Kinetics of substrate binding to human Cytochrome P450 3A4: A study with Fluorol‐7GA, a fluorescent allosteric ligand
Author(s) -
Rumfeldt Jessica Anne Osborne,
Halpert James R,
Davydov Dmitri R
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.512.5
Subject(s) - absorbance , chemistry , allosteric regulation , kinetics , fluorescence , heme , substrate (aquarium) , ligand (biochemistry) , steady state (chemistry) , analytical chemistry (journal) , conformational change , reaction rate constant , photochemistry , stereochemistry , chromatography , enzyme , biochemistry , physics , oceanography , receptor , quantum mechanics , geology
Stopped‐flow fluorescence and rapid scanning absorbance techniques were used to explore the mechanism of interactions of cytochrome P450 3A4 with Fluorol‐7GA, a fluorescent allosteric ligand. When monitored by rapid scanning absorbance spectroscopy, mixing of the enzyme and substrate resulted in a single exponential increase in the high spin fraction of the heme protein with a rate constant of ~10 s −1 . A lack of effect of the substrate concentration on the rate constant indicates that the observed process does not reflect the formation of the enzyme‐substrate complex per se but rather some transition in the pre‐formed complex that results in the displacement of the spin equilibrium. Stopped‐flow experiments, which monitored the intensity of fluorescence of Fluorol‐7GA, showed that the intensity decrease observed in steady state measurements takes place in the dead time of the instrument (≤2ms), and its kinetics are therefore too fast to be observed with our technique. Similar to the binding mechanism proposed from steady state measurements, the different results observed by stopped‐flow absorbance and fluorescence indicate a multistep binding process, namely initial very fast association followed by a first order structural transition that affects the spin state of the heme protein. (Supported by NIH grant GM054995).