COOPERATIVITY MECHANISM IN CYTOCHROME P450 3A4 DEMONSTRATED BY FRET AND ABSORBANCE SPECTROSCOPY

Abstract

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB) and 1-pyrenemethylamine (1-PMA) by FRET from the substrate fluorophore to the heme, as well as by absorbance spectroscopy. These approaches combined with dilution experiments performed at various enzyme-to-substrate ratios allowed us to resolve two substrate binding sites for 1-PB, and to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or the Type-II spectral changes caused by 1-PMA. Interaction of 3A4 with 1-PB reveals prominent homotropic cooperativity characterized by a Hill coefficient of 1.8 ± 0.3 (S50 = 8.0 ± 1.1 μM) when monitored by the spin shift in titration experiments. In contrast, the binding of 1-PMA results in Type II spectral changes, which obey a simple binary association equation (KD = 6.7 ± 1.9 μM) exhibiting no cooperativity. The use of FRET in dilution experiments at a 1:1 enzyme-to-substrate ratio revealed a binding site characterized by a KD of 0.2 ± 0.1 and 4.7 ± 1.4 μM for 1-PB and 1-PMA, respectively. Monitoring of the absorbance spectra in the dilution experiments showed that the high-affinity interactions with 1-PB do not cause any changes in the spin equilibrium of 3A4. Thus, 3A4 possesses two binding sites for 1-PB and one binding site for 1-PMA. The positive homotropic cooperativity with 1-PB results from the requirement that both sites be saturated for the spin transition to occur. (Supported by NIH grant GM54995 and Center Grant ES06676).