Abstract
Human cytochrome P450 (P450) 11B2 catalyzes the formation of aldosterone, the major endogenous human mineralocorticoid. Aldosterone is important for the regulation of electrolyte homeostasis. Mutations and overexpression of P450 11B2 (also known as aldosterone synthase) can lead to hypertension, congestive heart failure, and diabetic nephropathy. The enzyme is therefore a target for drug development to manage these various disorders. P450 11B2 catalyzes aldosterone formation from 11-deoxycorticosterone through three distinct oxidation steps. It is currently unknown to which degree these reactions happen in sequence without the intermediate products dissociating from the enzyme (i.e. processively) or whether these reactions happen solely distributively, in which the intermediate products must first dissociate and then rebind to the enzyme before subsequent oxidation. We present here a comprehensive investigation of processivity in P450 11B2-catalyzed reactions using steady-state, pre-steady-state, pulse-chase, equilibrium-binding titrations, and stopped-flow binding studies. We utilized the data obtained to develop a kinetic model for P450 11B2 and tested this model by enzyme kinetics simulations. We found that although aldosterone is produced processively, the enzyme preferentially utilizes a distributive mechanism that ends with the production of 18-OH corticosterone. This seemingly contradictory observation could be resolved by considering the ability of the intermediate product 18-OH corticosterone to exist as a lactol form, with the equilibrium favoring the ring-closed lactol configuration. In summary, our refined model for P450 11B2 catalysis indicates isomerization of the intermediate to a lactol can explain why P450 11B2 must produce aldosterone through a processive mechanism despite favoring a distributive mechanism.
Highlights
Bovine Adx and AdR were expressed in Escherichia coli DH5␣ cells (Invitrogen) and purified as described previously
Our results indicate slower rate values than that of Hobler et al [12], but a direct comparison is difficult in that their reported value was generated from all P450 11B2 reactions studied simultaneously, i.e. substrate disappearance
Each of the previously published studies reports that P450 11B2 cannot produce aldosterone directly from 18-OH corticosterone [11, 12, 21]
Summary
P450, cytochrome P450; Adx, adrenodoxin; AdR, NADPH–Adx reductase; DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; OH, hydroxy; Ni-NTA, nickel-nitrilotriacetic acid. P450 11B1 is primarily responsible for the synthesis of cortisol from deoxycortisol This reaction is an 11-hydroxylation, like the first step of aldosterone production by P450 11B2. It is responsible for both cortisol and aldosterone production Mechanistic studies on this system are likely relevant to understanding human P450 11B2 action [20]. Studies with human P450 11B2 from other laboratories have indicated that the enzyme catalyzes its reactions with varied reaction rates and specificities [11, 12, 21]. These studies each focused on different aspects of human P450 11B2 reactivity. Our steady-state and pre-steady–state results presented here agree with a number of previous reports and revealed additional insights regarding processivity, rate-limiting steps, and the role of chemical equilibration of a key intermediate with its lactol form
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