Regulation of 17,20 Lyase Activity by Cytochrome b5 and by Serine Phosphorylation of P450c17

Walter L. Miller,Amit V. Pandey

doi:10.1074/jbc.m414673200

Walter L. Miller, Amit V. Pandey

Open Access

https://doi.org/10.1074/jbc.m414673200

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Abstract

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.

Highlights

Cytochrome P450c17 is an essential steroid biosynthetic enzyme required for reproduction in all vertebrates
We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact
In some P450-mediated drug metabolism reactions, cytochrome b5 appears to act as an alternative electron donor that can substitute for P450 oxidoreductase (POR) in the donation of second electron in the P450 cycle (30 –32) but it does not function in this fashion to foster 17,20 lyase activity, as apo b5, which is devoid of heme, is as effective as holo b5 [13, 32]

Summary

Introduction

Cytochrome P450c17 is an essential steroid biosynthetic enzyme required for reproduction in all vertebrates. The ratio of 17,20 lyase activity to 17␣-hydroxylase activity can be regulated by three distinct post-translational mechanisms. 17,20 lyase activity can be enhanced by the presence of cytochrome b5 (24 –26), which acts allosterically to foster interactions with POR [13, 27]. In some P450-mediated drug metabolism reactions, cytochrome b5 appears to act as an alternative electron donor that can substitute for POR in the donation of second electron in the P450 cycle (30 –32) but it does not function in this fashion to foster 17,20 lyase activity, as apo b5, which is devoid of heme, is as effective as holo b5 [13, 32]. Regulation of 17,20 Lyase Activity show that these two mechanisms of augmenting 17,20 lyase activity function independently

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