Cytochrome P450
111
andinsertedintothemitochondrialmem-
brane before Fnal folding occurs. There is
no sequence homology of this extension
between different mitochondrial P450s
and its length varies signiFcantly from
one protein to another. Like other pre-
cursor sequences, however, mitochondrial
P450 precursor segments contain charged
amino acids. All membrane-bound P450s
are proposed to have a similar mode
of association with the membrane via a
large hydrophilic domain anchored to the
lipid bilayer through several noncontigu-
ous portions of the polypeptide chain, with
some of the membrane-interacting areas
being common to both mitochondrial and
microsomal P450s. It has also been sug-
gested that in membrane-bound P450s the
substrate may enter directly from the lipid
bilayer, thus providing a rationale as to how
hydrophobic substrates can avoid traveling
through the aqueous cytosol.
Other than addition of protoheme, post-
translational modiFcation is not a frequent
event in P450s. There have been re-
ports that phosphorylation is important in
modifying the function of speciFc forms
of P450, but this has not been Frmly
established. Glycosylation of aromatase
cytochrome P450 is known to occur, al-
though this modiFcation apparently has
no effect on the activity of the enzyme. Gly-
cosylation of other P450s is not common.
Heme binding remains the one posttrans-
lational event occurring in all P450s. How
it occurs, however, is not at all understood.
Since the last step of heme biosynthe-
sis (iron insertion into protoporphyrin
IX) occurs in the mitochondrion, mito-
chondrial P450s might Fnd heme quite
readily in the matrix. How heme gets to
the endoplasmic reticulum for insertion
into P450s and other heme proteins is a
mystery.
5
Regulation of P450 Activities
While certain P450-dependent activities,
such as the lanosterol 14
α
-demethylase
step in cholesterol biosynthesis and that
of 27-hydroxylase in bile acid biosynthe-
sis, have important constitutive regulatory
components, other P450 activities are reg-
ulated by signal transduction pathways.
Much of this regulation is at the tran-
scriptional level. ±or example, peptide
hormones from the anterior pituitary reg-
ulate transcription of P450s involved in
steroid hormone biosynthesis. Adrenocor-
ticotropin (ACTH), via cAMP-dependent
pathways, controls the transcription of
genes in the adrenal cortex, encoding both
mitochondrial and microsomal P450s in
the steroid hormone biosynthetic path-
way, as well as related proteins such as
ferredoxin. ACTH also controls the avail-
ability of substrate (cholesterol) in the
inner mitochondrial membrane, thereby
having the dual effect of regulating enzyme
levels and substrate availability. As another
example, parathyroid hormone regulates
P450-dependent vitamin D
3
metabolism
via cAMP, presumably at the transcrip-
tional level.
When considering regulation of P450
activities, there are four different regula-
tory components to be evaluated: devel-
opment, tissue-speciFc, constitutive, and
inducible. Not all P450s are inducible,
but the other three factors of this mul-
tifactorial regulatory scheme probably ap-
ply to all eukaryotic P450s. We do not
yet have a good view of the multi-
factorial regulation of most P450 en-
zymes, the steroid hydroxylases being the
best understood. In addition to peptide
hormone–dependent regulation through
cAMP, developmental/tissue-speciFc reg-
ulation is dependent on an orphan nuclear
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