Investigation of Adrenodoxin Mediated Allosteric Effects in Cytochrome P450 Family 11 Enzymes

Abstract

Human cytochrome P450 (CYP) enzymes are a diverse group of heme monooxygenases with a variety of biological functions, including the generation of steroid hormones. The CYP11 family includes CYP11A1, CYP11B1, and CYP11B2, which play major roles in the human steroidogenic pathway. CYP11A1 is responsible for the initial step in the steroidogenic pathway, the conversion of cholesterol to pregnenolone, while the CYP11B enzymes synthesize human corticosteroids. CYP11B1 synthesizes the major glucocorticoid cortisol, which plays a role in the stress response, whereas CYP11B2 synthesizes the major human mineralocorticoid aldosterone, which is crucial in salt/water balance. The CYP11 enzymes are drug targets for different disease states: metastatic prostate cancer (CYP11A1), Cushing syndrome (CYP11B1) and primary aldosteronism (CYP11B2). Targeting CYP11A1 is a novel therapeutic approach, whereas inhibitor development for CYP11B1 and CYP11B2 has been ongoing for many years but has been impeded by their 93% sequence identity. Further information is needed to identify differences between CYP11B1 and CYP11B2 enzymatic function that can be exploited to develop selective drugs for either disease state. One avenue to define functional differences includes the interaction with the common redox partner adrenodoxin, which is hypothesized to have an allosteric effect on the CYP11B enzymes in addition to its role as an electron donor. Herein, we determined that adrenodoxin is an allosteric modulator of both CYP11B enzymes, primarily through inducing a conformational change that enhances ligand binding. In equilibrium binding studies, adrenodoxin increases both substrate saturation and affinity, and in steady-state kinetic studies, increasing adrenodoxin concentrations decrease the apparent Km. Stopped-flow studies reveal that adrenodoxin increases substrate affinity for both CYP11B enzymes through a two-pronged effect: accelerating binding and decelerating release. This dual influence supports a complex and dynamic CYP11B conformational change upon adrenodoxin binding. In addition, these stopped-flow studies reveal important differences between the CYP11B enzymes: substrate binding is monophasic for CYP11B1 and multiphasic for CYP11B2, indicating complex mechanistic differences. While CYP11B1 follows a simple two-step substrate binding mechanism, kinetic modeling experiments support that CYP11B2 follows a more complex binding mechanism. In the absence of adrenodoxin, CYP11B2 substrate binding follows a 4-state induced fit model, and the addition of adrenodoxin shifts the binding mechanism to a 4-state closed model with combined conformational selection and induced fit steps. As the concentration of adrenodoxin increases, the rate constant governing the CYP11B2 conformational change also increases. This suggests that adrenodoxin promotes an CYP11B2 conformational change that enhances substrate binding. Adrenodoxin is also a common redox partner for CYP11A1, and adding this enzyme to the study provides important corollary information. Herein, we determined that CYP11A1 substrate binding also primarily follows a 4-state closed model, and increasing the concentration of adrenodoxin also causes the rate constant governing the conformational change to increase. Overall, this dissertation has investigated and characterized the interactions between the CYP11 enzymes, substrate, and adrenodoxin to better understand how adrenodoxin alters enzymatic function.