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

Abstract

Edited by Ruma Banerjee 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 17␣-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 17␣-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 b 5 partially enhances P450 17A1 lyase activity by altering the P450 17A1 conformation but did not measurably alter the binding of 17␣-OH pregnenolone or 17␣-OH progesterone, as judged by the apparent K d 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. Cytochrome P450 (P450) 2 enzymes are the main catalysts involved in the oxidation of steroids, drugs, fat-soluble vitamins, chemical carcinogens, and many other chemicals (1, 2). Collectively, they account for Ͼ95% of the oxidations and This work was supported by National Institutes of Health Grants R01 GM118122 (to F. P. G.) and T32 ES007028 (to F. P. G., support of S. M. G. and M. J. R.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article was selected as one of our Editors' Picks. This article contains Scheme S1 and Figs. S1-S8.

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

“…Several human P450s were examined regarding the kinetics of interaction with substrates, with the aim of developing models that are as simple as possible and judging whether inducedfit or conformational-selection dominates. Based on previous work (32,33,52) and new experimental studies, we conclude (Table 2) that (i) some systems are simple and can be represented by two states, (ii) most P450-substrate systems can be described by a conformational-selection model, (iii) some P450-substrate systems may be described by an induced-fit or a conformational-selection mechanism in the absence of more data, and (iv) some P450-substrate systems are still more complex and probably involve elements of both induced-fit and conformational-selection. The simple systems (item i and Table 2) may prove to be more complex upon further analysis.…”

“…We have analyzed P450s with multiple substrates ( Table 2) and concluded that conformational-selection was the dominant model in each case, as we did with P450 17A1 and seven steroids (52). In the case of P450 3A4 we analyzed data with four ligands (Figs.…”

“…4 All microsomal P450s bind NADPH-P450 reductase and some also bind cytochrome b 5 . With human P450 17A1, which is known to interact with cytochrome b 5 (86), we found that cytochrome b 5 did not modify the binding of the substrate 17␣-hydroxypregnenolone (52). No effect of NADPH-P450 reductase was seen in binding of substrates to P450s 1A2, 2A6, 2D6, and 3A4 (87).…”

“…We investigated the binding of steroids to human P450 17A1 and concluded that the mechanism is dominated by conformational-selection, not induced-fit (52). The conclusion was based upon (i) the decreasing rates of ligand binding as a function of steroid concentration (37), (ii) the differing plots of concentration dependence seen with the ligand and the enzyme (53), and (iii) comparisons made by fitting into KinTek Explorer models (54).…”

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

“…Proteins have the inherent ability to adopt a variety of thermally accessible conformational states, which play a key role in protein evolvability and activity 12,13 . Along the catalytic cycle, enzymes can adopt multiple conformations important for substrate binding or product release 14,15 , and conformational change can be rate-limiting in some cases 16,17 .…”