Analysis of Heterotropic Cooperativity in Cytochrome P450 3A4 Using α-Naphthoflavone and Testosterone

Abstract

Cytochrome P450 3A4 (CYP3A4) displays non-MichaelisMenten kinetics for many of the substrates it metabolizes, including testosterone (TST) and ␣-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.Hepatic cytochromes P450 play a fundamental role in the breakdown of xenobiotics from the blood. The most abundant of these in the adult human liver is cytochrome P450 3A4 (CYP3A4) 2 (1), which metabolizes approximately half of the most commonly prescribed drugs (2). Its ability to interact simultaneously with multiple substrate molecules leads to atypical kinetic phenomena, termed homotropic or heterotropic cooperativity (3-6), the former describing interactions of molecules of the same substrate and the latter referring to interactions of different substrates. Evidence of CYP3A4 simultaneously interacting with multiple substrate molecules comes from the crystal structures of the enzyme, which show a large, plastic active site (7), substrate bound at a peripheral binding site (8), changes in kinetic behavior in the presence of so-called effector molecules (9 -12), as well as a global analysis of multiple observable enzyme properties (13)(14)(15)(16).Recent work from our laboratory has shown that the apparent heterotropic cooperativity in Type I spin transition between ANF and TST can be accounted for by the additive effect of the ability of each substrate to induce the spin transition (17). Differences in their relative spectral affinities may give the appearance of a stimulatory effect of ANF on TST-induced spin transition; however, this is not indicative of any true cooperative behavior in binding (17). Here, we extend this analysis to include the contributions of NADPH oxidation and product-forming rates from each of the substrates to elucidate a more complete understanding of the heterotropic interactions of this important drug-metabolizing enzyme.

Drug–Drug Interactions between Atorvastatin and Dronedarone Mediated by Monomeric CYP3A4

“…Incorporation of CPR into preformed and purified CYP3A4-Nanodiscs was made by direct addition of CPR at 1:4 CYP3A4/CPR molar ratio, as described (48). All experiments were performed at 37° C using the POPC Nanodisc system similar to our earlier studies (34, 35), in order to allow direct comparison of results. This reconstitution system provides a stable, well characterized and monodisperse preparation of CYP3A4 incorporated into the model lipid bilayer effectively mimicking the native membrane.…”

“…All currently available experimental results on heterotropic activation of human cytochromes P450 suggest a specific allosteric response to steroids and other effectors having similar molecular properties, i.e. flat hydrophobic molecules (1, 34, 35). However, little is known about ability of other classes of substrates to interact with the same allosteric site and about possible effects of such interactions on the metabolism of clinically relevant substrates.…”

Membrane-embedded substrate recognition by cytochrome P450 3A4

“…(24) Using the CYP3A4 embedded in POPC nanodiscs, occupation of the high-affinity site induces only a minor shift in the iron spin state (22%) and under catalytic conditions NADPH oxidation is increased several fold without coupled product formation. Both of these observations are consistent with a TST binding site that is too far from the heme iron to efficiently displace the Fecoordinated water or undergo hydrogen-atom abstraction by the Compound I oxoferryl species.…”

“…The overwhelming majority of biophysical studies of CYP3A4-ligand interactions in model membranes have been performed with this version of the protein so it was adopted here to avoid confounding comparisons between simulation predictions and past experimental data. (24,(45)(46)(47)(48)(49)(50)(51) Protonation states of amino acids were assigned using Poisson-Boltzmann based PropKa calculations at pH 7.4.(52). Curated CYP3A4 was then inserted into a 75 Å 2 POPC membrane constructed using the Membrane Builder plugin of VMD.…”

“…The positive homotropic cooperativity in the oxidation of TST by CYP3A4 is well documented, and has been attributed to three TST binding sites. (13,24,43) The TST-CYP3A4 pair an ideal system to capture the structural details of these aspects of cytochrome P450 activity by simulation. aMD frequently and reproducibly identified binding sites in close proximity to the heme that are consistent with the partial heme spin-shift and NADPH oxidation without coupled product formation previously observed at low TST concentrations.…”