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  • For both the hydroxylation and lyase reactions the heme

    2023-11-29

    For both, the 17α-hydroxylation and 17, 20-lyase reactions, the heme iron center of P450 17A1 receives electron reducing equivalents from CPR. Both reactions require one pair of electrons and molecular oxygen for the conversion of the substrate into product in each catalytic cycle. However, the 17,20-lyase reaction has been proposed to use a ferric peroxide (Fe(II)O2–) rather than the Obeticholic Acid I (Cpd I) that normally is used in P450 reactions [155], [156], [157]. It has been reported that the cyt b5 and phosphorylation of P450 17A1 can only stimulate the lyase reaction and not the 17α-hydroxylation reaction [99], [158], [159], [160], [161]. Recent NMR studies have revealed that the human P450 17A1 has the same binding site for both CPR as for cyt b5 [109], however, unlike CPR the cyt b5 stimulates the lyase reaction without transferring electrons [162]. Furthermore, a peroxo-hemiacetal pregnenolone intermediate has been recently identified using low temperature Raman spectroscopy, during 17,20-lyase activity [270]. During the catalytic reaction of bovine P450 17A1, the intermediates have been shown not to dissociate from the multi-enzyme complex before the next step [163], [164], [165], [166], [167], [168], [169], [170]. The P450 17A1 catalyzed reactions also vary in some other points, depending upon the animal species [154] and among the different species the major differences lie in the lyase activity and hydroxylated intermediates [171]. This is discussed further in section 3.2. In addition to 17α-hydroxylation activity for the human P450 17A1, the substrate progesterone can also be 16α-hydroxylated [158]. Swart et al. measured the ratio between the 16α-hydroxy-:17α-hydroxy- progesterone as 1:2 and 1:4. The Km values for these 16α- and 17α-hydroxylations are almost the same, although the Vmax of the P450 17A1 for 17α-hydoxyprogesterone production is about 2-times higher than that for the 16α-hydroxyprogesterone production [172], [173], [174]. Even though the specific role of P450 17A1 to 16-hydroxyprogesterone is still not clear, these workers demonstrated that across a range of mammals the ability to hydroxylate progesterone at C-16 is linked to a single amino acid residue, Ala105 [174], [175]. Interestingly, in vivo humans share this Ala105 with chimpanzees but not with baboon, caprine (goat), porcine [174] or bovine [175] species which all have a leucine at position 105. Comparing these species and enzymes constructed with Ala105 or Leu105, Ala at position 105 clearly skews the activity towards 16-hydoxylation (Fig. 4 in [174]). However there was also an apparent reduction in activity when L105A was constructed from the baboon, goat and pig, based upon our interpretation of Fig. 3 in [174] where the mutant has resulted in less metabolism of the substrate progesterone. It was suggested that the 16α-hydroxylation reaction occurs in human and chimpanzee P450 17A1 as the Ala105 amino acid residue is located in the B́-C domain is small and provides sufficient space to position the progesterone for the 16α-hydroxylation reaction, as illustrated by the recent X-ray structure of this mutant [176]. However, in other species (baboon, goat and pig) the Ala105 residue has been replaced by the larger leucine residue, which prevents the reposition of the substrate through steric-hindrance (Fig. 6). The presence of cyt b5 enhanced the 16α-hydroxylase activity of P450 17A1 in species that contained Leu105 (human Ala105Leu, wt baboon, wt caprine and wt porcine P450 17A1) [174]. Although, since the 16α-hydroxylase reaction does not require a second catalytic cycle. This 16α-hydroxylase activity has been attributed to the allosteric effect of cyt b5 changing the 3D conformation of P450 17A1 so that substrate could align with it in a proper orientation to favor the reaction [174]. The activity of mutated Lys105 to Ala105 in P450 17A1 (baboon, goat and pig) was not significantly affected by cyt b5. This suggested that the allosteric effect of cyt b5 did not enhance the binding of the substrate to P450 17A1. Notably, porcine P450 17A1 has been found to accept both 17α-hydroxypregnenolone and 17α-hydroxyprogesterone as substrates for the 17,20-lyase reaction [177], [178], [179].