Kinetic analysis of the three-step steroid aromatase reaction of human cytochrome P450 19A1

Abstract

Cytochrome P450 19A1 (P450 19A1), the aromatase, catalyzes the conversion of androgens to estrogens through a sequential three-step reaction, generating 19-hydroxy and 19-aldehyde intermediates en route to the product estrogen. A procedure for the heterologous expression and purification of P450 19A1 in Escherichia coli was developed (k(cat) of 0.06 s(-1) for the conversion of androstenedione to estrone). Binding of the substrate and intermediates show low micromolar dissociation constants and are at least two-step processes. Rates of reduction of the iron were fast in the presence of substrate, either intermediate, or product. P450 19A1 is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the intermediates as revealed by pulse-chase experiments. Conversion of androstenedione to estrone (under single turnover conditions) generated a progress curve showing changes in the concentrations of the substrate, intermediates, and product. A minimal kinetic model containing the individual rate constants for the steps in P450 19A1 catalysis was developed to globally fit the time course of the overall reaction, the dissociation constants, the two-step ligand binding, the distributive character, the iron-reduction rates, and the steady-state conversion of the 19-hydroxy androstenedione and 19-aldehyde androstenedione intermediates to estrone.

FIGURE 1.

General P450 catalytic cycle.

SCHEME 1.

FIGURE 2.

N-terminal sequence and purification of P450 19A1 protein. A, N-terminal sequence of native mammalian P450 19A1 (top) and the modification used for heterologous expression (bottom). B, SDS-PAGE (10% gel, stained with Coomassie Brilliant Blue G250). Lane 1, whole cells; lane 2, membrane-bound fraction; lane 3, solubilized membrane fraction; and lane 4, purified P450 19A1. Nominal protein concentrations loaded in lanes 1–4 were 70, 55, 7, and 1.7 μg, respectively.

FIGURE 3.

Spectra of purified P450 19A1. A, Fe²⁺·CO versus Fe²⁺ difference spectrum (using 2 μm safranin T dye as an electron mediator). B, absolute spectrum showing ferric and ferrous forms. C, absolute spectrum of 2 μm P450 19A1 in the absence (solid line) or presence (small dotted line) of 10 μm andro and the difference between the two spectra (large dotted line). Also shown (inset) are the second derivatives of the spectra recorded in the absence (solid line) or presence (large dotted line) of 10 μm andro, showing about 18% conversion from low- to high-spin iron upon ligand binding.

FIGURE 4.

Steady-state binding of ligands to P450 19A1. Titration of P450 19A1 (1 μm) with varying concentrations of ligand. Insets show the spectral changes, and the plots (lines) are quadratic fits of the changes in absorbance (squares). A, andro, K_d = 0.13 ± 0.07 μm; B, 19-OH andro, K_d = 1.5 ± 0.4 μm; C, 19=O andro, K_d = 3.6 ± 0.6 μm; and D, estrone, K_d = 4.0 ± 1.0 μm.

FIGURE 5.

Pre-steady-state binding kinetics of ligands to P450 19A1. Stopped-flow absorbance (A–C) and fluorescence (D) changes for binding of various ligands (2 μm) to P450 19A1 (2 μm). Raw data are presented as scatter plots, and the overlaid lines are fits using Dynafit software. A, andro (ΔA₃₉₀), k₁ = 2.5 × 10⁶ m⁻¹ s⁻¹, k₋₁ = 1.4 s⁻¹, k₂ = 0.42 s⁻¹, k₋₂ = 0.20 s⁻¹; B, 19-OH andro (ΔA₃₉₀), k₁ = 2.0 × 10⁷ m⁻¹ s⁻¹, k₋₁ = 240 s⁻¹, k₂ = 0.80 s⁻¹, k₋₂ = 0.15 s⁻¹; C, 19=O andro (ΔA₃₉₀), k₁ = 2.5 × 10⁶ m⁻¹ s⁻¹, k₋₁ = 300 s⁻¹, k₂ = 2.4 s⁻¹, k₋₂ = 0.13 s⁻¹; and D, αNF (ΔF_{> 385}), k₁ = 1.0 × 10⁷ m⁻¹ s⁻¹, k₋₁ = 0.1 s⁻¹, k₂ = 0.01 s⁻¹, k₋₂ = 0.18 s⁻¹.

FIGURE 6.

Reduction kinetics of P450 19A1. Stopped-flow absorbance traces of the reduction of P450 19A1 (1 μm) by NADPH-P450 reductase (2 μm) in the presence of l-α-1,2-dilauroyl-sn-glycero-3-phosphocholine (95 μm) and various ligands (5 μm) upon the addition of NADPH (150 μm). A, andro (ΔA₄₅₀), k = 1.7 ± 0.1 s⁻¹; B, 19-OH andro (ΔA₃₉₁), k_fast = 10 ± 4 s⁻¹, k_slow = 0.97 ± 0.12 s⁻¹; C, 19=O andro (ΔA₄₅₀), k_fast = 5.4 ± 0.5 s⁻¹, k_slow = 0.34 ± 0.06 s⁻¹; and D, estrone (ΔA₃₉₁), k_fast = 2.2 ± 0.4 s⁻¹, k_slow = 0.098 ± 0.02 s⁻¹. The analysis of residuals is shown at the top of each plot.

FIGURE 7.

Steady-state kinetics of P450 19A1 activity. Hyperbolic fitting (lines) of the formation of estrone by P450 19A1. A, andro as substrate; B, 19-OH andro as substrate; and C, 19=O andro as substrate. Data points are squares.

FIGURE 8.

Pulse-chase assays. P450 19A1 (2 nm) was incubated with 400 nm [1,2,6,7-³H]andro, followed by a pulse of 19-OH andro (210 μm) or 19=O andro (180 μm) at 30 or 60 s. In this case, 100% activity was 0.087 s⁻¹. The total reaction time was 5 min.

FIGURE 9.

Kinetics of P450 19A1 catalysis under single turnover conditions. Loss and/or formation of andro (red), 19-OH andro (orange), 19=O andro (green), and estrone (blue) were monitored in a reaction of P450 19A1 (2 μm) and [4-¹⁴C]andro (2 μm). Global fitting (using Kintek Explorer® software) is shown.