Kinetics of Intermediate Release Enhances P450 11B2-Catalyzed Aldosterone Synthesis

Abstract

The mitochondrial enzyme cytochrome P450 11B2 (aldosterone synthase) catalyzes the 3 terminal transformations in the biosynthesis of aldosterone from 11-deoxycorticosterone (DOC): 11β-hydroxylation to corticosterone, 18-hydroxylation, and 18-oxidation. Prior studies have shown that P450 11B2 produces more aldosterone from DOC than from the intermediate corticosterone and that the reaction sequence is processive, with intermediates remaining bound to the active site between oxygenation reactions. In contrast, P450 11B1 (11β-hydroxylase), which catalyzes the terminal step in cortisol biosynthesis, shares a 93% amino acid sequence identity with P450 11B2, converts DOC to corticosterone, but cannot synthesize aldosterone from DOC. The biochemical and biophysical properties of P450 11B2, which enable its unique 18-oxygenation activity and processivity, yet are not also represented in P450 11B1, remain unknown. To understand the mechanism of aldosterone biosynthesis, we introduced point mutations at residue 320, which partially exchange the activities of P450 11B1 and P450 11B2 (V320A and A320V, respectively). We then investigated NADPH coupling efficiencies, binding kinetics and affinities, and product formation of purified P450 11B1 and P450 11B2, wild-type, and residue 320 mutations in phospholipid vesicles and nanodiscs. Coupling efficiencies for the 18-hydroxylase reaction with corticosterone as the substrate failed to correlate with aldosterone synthesis, ruling out uncoupling as a relevant mechanism. Conversely, corticosterone dissociation rates correlated inversely with aldosterone production. We conclude that intermediate dissociation kinetics, not coupling efficiency, enable P450 11B2 to synthesize aldosterone via a processive mechanism. Our kinetic data also suggest that the binding of DOC to P450 11B enzymes occurs in at least two distinct steps, favoring an induced-fit mechanism.

Figure 1.

Reactions catalyzed by cytochrome P450 11B1 and P450 11B2 (Panel A) and interconversion between the open and lactol form of 18-hydroxycorticosterone (Panel B).

Figure 2.

Steady-state activities of wild-type and mutant P450 11B enzymes in phospholipid vesicles. Data represent the mean ± standard deviation of three experiments (data points) with <25% DOC consumption (100 μM);

Figure 3.

Equilibrium binding titrations of DOC and corticosterone to P450 11B enzymes in nanodiscs. Panels A, C, E, and G show DOC binding to P450 11B1 (□), P450 11B1-V320A (Δ), P450 11B2 (◇) and P450 11B2-A320V (○), respectively. Panels B, D, F, and H show corticosterone binding to the same enzymes. The insets in each panel show the difference spectra at each ligand concentration. Data represent the mean ± standard deviation of three experiments. Error bars in panels C and D are smaller than the symbols.

Figure 4.

UV-Visible stopped-flow analysis of P450 11B enzymes in nanodiscs. Panels A, B, C, and D show the binding of 0.25 μM of P450 11B1 (□), P450 11B1-V320A (Δ), P450 11B2 (◇) and P450 11B2-A320V (○), respectively, with DOC at the following concentrations: 1 μM (black), 2 μM (magenta), 4 μM (aqua), 8 μM (purple), and 16 μM (lilac). The lines in panels A-D show the nonlinear regression fits to the traces obtained, which represent the mean of three technical replicates. Panels E-H show residual plots for curve fits to panels A-D, respectively. Panels I, J, K, and L show the linear analysis for the fast phase of the exponential fits as a function of [DOC], and the dashed lines represent the 95% confidence intervals. Panels M, N, O, and P show the calculated observed rate constants for the slow phase as a function of [DOC].

Figure 5.

Stopped-flow analysis of DOC dissociation from P450 11B enzymes by direct method. Panels A, B, C, and D show the mixing of 0.5 μM of P450 11B2 (◇) and 11B2-A320V (○) and P450 11B1 (□), and 11B1-V320A (Δ), respectively, with an excess of cyclodextrin. The dashed lines represent the 95% confidence intervals, and the insets show residual plots.

Figure 6.

Active site structures of P450 11B1 (PDB code 6M7X) and P450 11B2 (PDB code 7M8V) without ligand. The I-helix is shown in blue, and the heme prosthetic group is presented in orange in all panels. Panel A shows the placement and location of the valine residue at position 320, as highlighted in red in P450 11B1 (green). Panel B shows the placement and location of the alanine residue at position 320, as highlighted in red in P450 11B2 (aquamarine). Panels C and D highlight interactions between the 320 residues in P450 11B1 and P450 11B2, respectively, and neighboring residues, particularly F321 and L324. A water molecule (magenta) is represented as a sphere in panel C.