CO Binding Kinetics of Human Cytochrome P450 3A4

Abstract

The kinetics of CO binding to human cytochrome P450 3A4 was examined by the flash photolysis technique, employing the membrane-bound P450 expressed in baculovirus-infected SF9 insect cells. Triexponential kinetics was observed, indicating that P450 3A4 is composed of multiple, kinetically distinguishable conformers. To define the substrate specificity of individual P450 3A4 conformers we evaluated the effect of a series of substrates of varying sizes and structures on the CO binding kinetics. The rate of CO binding to the total mixture of P450 3A4 conformers was increased in the presence of nifedipine and erythromycin, decreased by quinidine, testosterone, and warfarin, and unaffected by cimetidine and 17 alpha-ethynylestradiol. A recently developed kinetic difference method (Koley, A. P., Robinson, R. C., Markowitz, A., and Friedman, F. K. (1994) Biochemistry 33, 2484-2489) was used to define the kinetic parameters of individual P450 3A4 conformers. The results showed that different conformers have distinct substrate specificities. The substrates had markedly variable effects on the CO binding kinetics of their target P450 3A4 conformers and thus differentially modulate their conformations. These results demonstrate that the interaction of a particular substrate with a specific P450 3A4 conformer can be assessed in the presence of multiple conformers.

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

“…However, the addition of 1 -10 mM GSH results in an increased affinity of CYP3A4 for 1-PB and does not affect the S 50 values for the binding of BFC, testosterone, or ANF, suggesting that there is no competition between coordination of GSH to the heme iron and the interactions of CYP3A4 with these substrates. A possible explanation for this observation may be provided by the concept of persistent heterogeneity of CYP3A4 [37,38,[64][65][66] caused by dissimilarity of the orientation and/or conformation of the subunits in the oligomers (aggregates) in solution [38,66]. Lack of competition between GSH ligation to the heme and substrate binding may be caused by heterogeneity of oligomeric P450 in solution, similar to that revealed in barotropic behavior of CYP3A4 [37], kinetics of the enzyme reduction by dithionite [38], and CO-binding kinetics [64].…”

“…A possible explanation for this observation may be provided by the concept of persistent heterogeneity of CYP3A4 [37,38,[64][65][66] caused by dissimilarity of the orientation and/or conformation of the subunits in the oligomers (aggregates) in solution [38,66]. Lack of competition between GSH ligation to the heme and substrate binding may be caused by heterogeneity of oligomeric P450 in solution, similar to that revealed in barotropic behavior of CYP3A4 [37], kinetics of the enzyme reduction by dithionite [38], and CO-binding kinetics [64]. A suggestion that only some of the P450 molecules constituting the oligomer in solution have an orientation permitting them to bind GSH by ligation to the heme iron is consistent with our observation that, upon saturation of CYP3A4 with GSH the fraction of the enzyme represented by its thiol-and thiolate-ligated heme complexes does not exceed 40%.…”

Energetics of heterotropic cooperativity between α-naphthoflavone and testosterone binding to CYP3A4

“…There are three prevailing models of cooperativity for cytochrome P450s (CYPs): the multisubstrate binding site model [11][12][13], the peripheral effector binding site model [14][15][16], and the conformational heterogeneity model [17][18][19]. With the multi-substrate binding model, multiple substrates are bound at unproductive binding sites within the active site that can modulate the apparent K m , k cat and V max of substrate that transiently occupies a metabolically productive position.…”

Differential Mechanisms of Cytochrome P450 Inhibition and Activation by α-Naphthoflavone

“…Human P450s 3A4 and 1A1-These P450s were expressed in SF9 insect cells using recombinant baculoviruses (36, 37) and prepared for flash photolysis as described (34,35).…”

“…Thus, a higher rate of CO binding indicates a wide ligand access channel and/or a flexible protein, whereas a lower rate suggests a restricted access channel and/or rigid protein conformation. This kinetic approach was previously used to define P450-substrate interactions using both liver microsomes (32,33) and individual P450s (34,35).…”