The initial observations indicating a crucial
The initial observations indicating a crucial role for WZ811 transfer in 17,20 lyase activity were that the molar ratio of POR to P450c17 is three- to four-fold higher in porcine testes than in porcine adrenals, and that adding purified POR to porcine P450c17 in vitro increased 17,20 lyase more than 17-hydroxylase (Yanagibashi and Hall, 1986). This POR-dependency of the 17,20 lyase reaction was then confirmed in cell transfection experiments where an increased POR/P450c17 ratio led to more 17,20 lyase activity (Lin et al., 1993). Work with rodent, bovine and porcine P450c17 indicated that both 17-Preg and 17OHP were equally good substrates for the 17,20 lyase activity, or, in rodents, that the preferred substrate was 17OHP, in which case androgen synthesis could proceed through androstenedione without the intermediacy of DHEA. However, although human P450c17 catalyzes the 17α-hydroxylation of both pregnenolone and progesterone with approximately equal efficiency (Auchus et al, 1998, Lee-Robichaud et al, 1995), it catalyzes the 17,20 lyase reaction using 17-Preg as its principal substrate (Lin et al, 1991, Lin et al, 1993), with a 50:1 preference for 17-Preg over 17OHP (Auchus et al., 1998). Thus human P450c17 converts 17OHP to androstenedione only when the concentrations of 17OHP are extraordinarily high, as in 21-hydroxylase deficiency.
Structure of P450c17 To understand how P450c17 catalyzes its activities, we constructed a computational model and tested it by structure analysis programs, energetic constraints and molecular dynamics with and without each substrate docked in the active site (Auchus and Miller, 1999). The predicted active site accommodated substrate in a catalytically favorable orientation and the predicted contributions of positively charged residues to the redox-partner binding site were confirmed by site-directed mutagenesis. The model explained the activities of all known human P450c17 mutants, and correctly predicted that the mutation K89N would preferentially disrupt 17,20-lyase activity. Biochemical data suggested that both the hydroxylase and lyase activities proceed by an iron oxene mechanism. Crystallographic structures of human P450c17 with the inhibitors abiraterone or galeterone bound in the active site largely confirm the model (DeVore and Scott, 2012). However, whereas the model predicts that the substrate binds with the steroid parallel to the heme ring, with the α-surface of the D-ring nearest the heme iron, the X-ray structures show the heterocycle nitrogen of the inhibitors tightly bound to the heme iron and the 3β-face of the steroid forming hydrophobic interactions with the I-helix. Modeling pregnenolone into the space occupied by abiraterone places the H-17 atom in close proximity to the heme iron (DeVore and Scott, 2012). Pregnenolone carbon 21 is closer to the heme iron (2.9 Å) than is carbon 17 (3.6 Å), yet 17α-hydroxylation is kinetically much more favorable (Mizrachi et al, 2011, Yoshimoto et al, 2012). Solution NMR studies show that P450c17 undergoes multiple backbone conformations influenced by temperature, substrate binding, and binding of the soluble domain of cytochrome b5. When cytochrome b5 is added to P450c17 with pregnenolone bound in the P450c17 active site, the cytochrome b5 induces a P450c17 conformation that is similar to that seen in P450c17 with 17-Preg in its active site; this is consistent with the ability of cytochrome b5 to enhance 17,20-lyase activity (Estrada et al., 2014).
Human mutations that illuminate 17,20 lyase activity Disorders of human steroidogenesis that selectively impair 17,20 lyase activity have provided key insights into this activity (reviewed by Miller, 2012). The first cases of selective 17,20 lyase deficiency characterized by mutation analysis and enzymology were found in the redox-partner binding site of P450c17 from two 46,XY patients who had normal basal (but ACTH-unresponsive) cortisol, low C19 steroids and genital ambiguity (Geller et al., 1997). One patient was homozygous for the mutation R347H and the other was homozygous for R358Q; computational modeling showed that both mutated residues change the surface charge distribution in the redox-partner binding site of P450c17. Both mutations selectively impaired 17,20 lyase activity in transfected cells, but had minimal effect on 17α-hydroxylase activity (Geller et al., 1997). Assays in a yeast microsome system show that these mutant forms of P450c17 had the same Michaelis constant (Km) as the wild-type enzyme, but had reduced maximum velocities (Vmax) for the 17,20 lyase reaction and were subject to competitive inhibition, confirming that the mutations did not affect the active site (Geller et al., 1999). This impairment in 17,20 lyase activity could be partially corrected by a molar excess of cytochrome b5, providing evidence for the role of cytochrome b5 as an allosteric factor promoting the interaction of P450c17 with POR (Geller et al., 1999). Subsequent studies found additional patients with R347H and with the similar mutation R347C, which caused an indistinguishable phenotype and also had selective loss of 17,20 lyase activity in vitro (Kate-Booij et al, 2004, VanDenAkker et al, 2002).