Analysis of heterotropic cooperativity in cytochrome P450 3A4 using α-Naphthoflavone and testosterone

Daniel J. Frank, Ilia G. Denisov, Stephen G. Sligar

Research output: Contribution to journalArticle

Abstract

Cytochrome P450 3A4 (CYP3A4) displays non-Michaelis-Menten kinetics for many of the substrates it metabolizes, including testosterone (TST) and α-naphthoflavone (ANF). Heterotropic effects between these two substrates can further complicate the metabolic profile of the enzyme. In this work, monomeric CYP3A4 solubilized in Nanodiscs has been studied for its ability to interact with varying molar ratios of ANF and TST. Comparison of the observed heme spin state, NADPH consumption, and product formation rates with a non-cooperative model calculated from a linear combination of the global analysis of each substrate reveals a detailed landscape of the heterotropic interactions and indicates negligible binding cooperativity between ANF and TST. The observed effect of ANF on the kinetics of TST metabolism is due to the additive action of the second substrate with no specific allosteric effects.

Original languageEnglish (US)
Pages (from-to)5540-5545
Number of pages6
JournalJournal of Biological Chemistry
Volume286
Issue number7
DOIs
StatePublished - Feb 18 2011

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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