Characterization of human adrenal cytochrome P450 11B2 products of progesterone and androstenedione oxidation.

Cytochrome P450 (P450) 11B1 and 11B2 both catalyze the 11ß-hydroxylation of 11-deoxycorticosterone and the subsequent 18-hydroxylation of the product. P450 11B2, but not P450 11B1, catalyzes a further C-18 oxidation to yield aldosterone. 11-Oxygenated androgens are of interest, and 11-hydroxy progesterone has been reported to be a precursor of these. Oxidation of progesterone by purified recombinant P450 11B2 yielded a mono-hydroxy derivative as the major product, and co-chromatography with commercial standards and 2-D NMR spectroscopy indicated 11ß-hydroxylation. 18-Hydroxyprogesterone and a dihydroxyprogesterone were also formed. Similarly, oxidation of androstenedione by P450 11B2 yielded 11ß-hydroxyandrostenedione, 18-hydroxyandrostenedione, and a dihydroxyandrostenedione. The steady-state kinetic parameters for androstenedione and progesterone 11ß-hydroxylation were similar to those reported for the classic substrate 11-deoxycorticosterone. The source of 11α-hydroxyprogesterone in humans remains unresolved.

In Vitro Activation of Cytochrome P450 46A1 (CYP46A1) by Efavirenz-Related Compounds.

Cytochrome P450 46A1 (CYP46A1) is a central nervous system-specific enzyme, which catalyzes cholesterol 24-hydroxylation. Currently CYP46A1 is being evaluated in a clinical trial for activation by small doses of the anti-HIV drug efavirenz. Eight efavirenz-related compounds were investigated for CYP46A1 activation in vitro, induction of a CYP46A1 spectral response, spectral Kd values, interaction with the P450 allosteric sites, and a model of binding to the enzyme active site. We gained insight into structure-activity relationships of efavirenz for CYP46A1 activation and found that the investigated efavirenz primary metabolites are stronger and better activators of CYP46A1 than efavirenz. We also established that CYP46A1 is activated by racemates and that a conformational-selection mechanism is operative in CYP46A1. The results suggest structural modifications of efavirenz to further increase CYP46A1 activation without inhibition at high compound concentrations. It is possible that not only efavirenz but its metabolites activate CYP46A1 in vivo.

Human cytochrome P450 enzymes bind drugs and other substrates mainly through conformational-selection modes.

Cytochrome P450 (P450) enzymes are major catalysts involved in the oxidations of most drugs, steroids, carcinogens, fat-soluble vitamins, and natural products. The binding of substrates to some of the 57 human P450s and other mammalian P450s is more complex than a two-state system and has been proposed to involve mechanisms such as multiple ligand occupancy, induced-fit, and conformational-selection. Here, we used kinetic analysis of binding with multiple concentrations of substrates and computational modeling of these data to discern possible binding modes of several human P450s. We observed that P450 2D6 binds its ligand rolapitant in a mechanism involving conformational-selection. P450 4A11 bound the substrate lauric acid via conformational-selection, as did P450 2C8 with palmitic acid. Binding of the steroid progesterone to P450 21A2 was also best described by a conformational-selection model. Hexyl isonicotinate binding to P450 2E1 could be described by either a conformational-selection or an induced-fit model. Simulation of the binding of the ligands midazolam, bromocriptine, testosterone, and ketoconazole to P450 3A4 was consistent with an induced-fit or a conformational-selection model, but the concentration dependence of binding rates for varying both P450 3A4 and midazolam concentrations revealed discordance in the parameters, indicative of conformational-selection. Binding of the P450s 2C8, 2D6, 3A4, 4A11, and 21A2 was best described by conformational-selection, and P450 2E1 appeared to fit either mode. These findings highlight the complexity of human P450-substrate interactions and that conformational-selection is a dominant feature of many of these interactions.

Conformational selection dominates binding of steroids to human cytochrome P450 17A1.

Cytochrome P450 (P450, CYP) enzymes are the major catalysts involved in the oxidation of steroids as well as many other compounds. Their versatility has been explained in part by flexibility of the proteins and complexity of the binding mechanisms. However, whether these proteins bind their substrates via induced fit or conformational selection is not understood. P450 17A1 has a major role in steroidogenesis, catalyzing the two-step oxidations of progesterone and pregnenolone to androstenedione and dehydroepiandrosterone, respectively, via 17α-hydroxy (OH) intermediates. We examined the interaction of P450 17A1 with its steroid substrates by analyzing progress curves (UV-visible spectroscopy), revealing that the rates of binding of any of these substrates decreased with increasing substrate concentration, a hallmark of conformational selection. Further, when the concentration of 17α-OH pregnenolone was held constant and the P450 concentration increased, the binding rate increased, and such opposite patterns are also diagnostic of conformational selection. Kinetic simulation modeling was also more consistent with conformational selection than with an induced-fit mechanism. Cytochrome b5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17α-OH pregnenolone or 17α-OH progesterone, as judged by the apparent Kd and binding kinetics. The P450 17A1 inhibitor abiraterone also bound to P450 17A1 in a multistep manner, and modeling indicated that the selective inhibition of the two P450 17A1 steps by the drug orteronel can be rationalized only by a multiple-conformation model. In conclusion, P450 17A1 binds its steroid substrates via conformational selection.

Glutamine-451 Confers Sensitivity to Oxidative Inhibition and Heme-Thiolate Sulfenylation of Cytochrome P450 4B1.

Human cytochrome P450 (P450) family 4 enzymes are involved in the metabolism of fatty acids and the bioactivation of carcinogenic arylamines and toxic natural products, e.g., 4-ipomeanol. These and other drug-metabolizing P450s are redox sensitive, showing a loss of activity resulting from preincubation with H2O2 and recovery with mild reducing agents [Albertolle, M. W., et al. (2017) J. Biol. Chem. 292, 11230-11242]. The inhibition is due to sulfenylation of the heme-thiolate ligand, as determined by chemopreoteomics and spectroscopy. This phenomenon may have implications for chemical toxicity and observed disease-drug interactions, in which the decreased metabolism of P450 substrates occurs in patients with inflammatory diseases (e.g., influenza and autoimmunity). Human P450 1A2 was determined to be redox insensitive. To determine the mechanism underlying the differential redox sensitivity, molecular dynamics (MD) simulations were employed using the crystal structure of rabbit P450 4B1 (Protein Data Bank entry 5T6Q ). In simulating either the thiolate (Cys-S-) or the sulfenic acid (Cys-SOH) at the heme ligation site, MD revealed Gln-451 in either an "open" or "closed" conformation, respectively, between the cytosol and heme-thiolate cysteine. Mutation to either an isosteric leucine (Q451L) or glutamate (Q451E) abrogated the redox sensitivity, suggesting that this "open" conformation allows for reduction of the sulfenic acid and religation of the thiolate to the heme iron. In summary, MD simulations suggest that Gln-451 in P450 4B1 adopts conformations that may stabilize and protect the heme-thiolate sulfenic acid; mutating this residue destabilizes the interaction, producing a redox insensitive enzyme.