In addition
to the hydroxylation of aromatic amino acids, BH4 serves
as the cofactor for nitric oxide synthase2 and
glyceryl-ether monooxygenase3 (Figure
1).
Aromatic
Amino Acid Hydroxylases
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Figure 1

The function of these reactions derives from
the ability of BH4 to react with molecular oxygen to form
an active oxygen intermediate that can hydroxylate substrates.
In the hydroxylation process, the co-enzyme loses two electrons
and is regenerated in vivo in an NADH-dependent reaction.
Although BH4 was found to be absolutely essential for nitric
oxide synthase activity, the exact function in different
forms of the enzyme and the mechanism of action are as
yet not clear. Furthermore, it has been demonstrated that
BH4 increases the proliferation rate of erythroid cells4 and
that it is active as a dopamine releasing factor in rat
striatum by using an in vivo dialysis technique5.
Metabolism
of tetrahydrobiopterin
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BH4 is synthesized from guanosine triphosphate
(GTP) in at least four enzymatic steps by the action of
three different enzymes (see Figure
1)6-9.
GTP cyclohydrolase I (GTPCH), the first enzyme in BH4 biosynthesis,
catalyzes the formation of 7,8-dihydroneopterin triphosphate
from GTP in a single reaction step10.
GTPCH is subject to feed-back inhibition by BH4. Inhibition
occurs through a BH4-dependent complex formation between
a newly discovered p35 protein and GTPCH11.
Furthermore, the inhibition is specifically reversed by
phenylalanine. These may explain the high neopterin and
biopterin content observed in patients with HPA12.

In the following step, the 6-pyruvoyl-tetrahydropterin
synthase (PTPS) catalyzes the conversion of 7,8-dihydroneopterin
triphosphate to 6-pyruvoyl-tetrahydropterin13.
This conversion is magnesium and zinc dependent and involves
the elimination of triphosphate and an intermolecular redox
rearrangement reaction14.
Whereas GTPCH is found to be highly regulated by the p35
protein, for PTPS no such regulatory processes are described.
Nevertheless, PTPS needs posttranslational modification(s),
i.e. phosphorylation, in order to be fully active in vivo15.
Furthermore, it has been suggested that PTPS is the rate
limiting enzyme for BH4 biosynthesis, at least in human
liver16

Sepiapterin reductase (SR) has been shown
to be an NADPH oxidoreductase required for the final two-step
reduction of the diketo intermediate 6-pyruvoyl-tetrahydropterin
to BH417.

During the enzymatic hydroxylation of aromatic
amino acids, molecular oxygen is consumed and tetrahydrobiopterin
is peroxidated and oxidized. The pterin intermediate is
subsequently reduced back to BH4 by two enzymes and a reduced
pyridine nucleotide (NADH) in a complex recycling reaction
(Figure
1). Molecular oxygen is first bound to BH4 to form
an unstable 4a-peroxy-tetrahydrobiopterin. The monooxygenation
of aromatic amino acids is thus concomitant with oxidation
of BH4 to 4a-hydroxy-tetrahydrobiopterin (pterin-4a-carbinolamine)18.
Pterin-4a-carbinolamine is subsequently dehydrated to quinonoid-dihydrobiopterin
(q-dihydrobiopterin) and water by the specific and highly
efficient pterin-4a-carbinolamine dehydratase (PCD)19,
20.

The PCD was originally detected as a contaminant in a preparation of
the rat PAH as a consequence of its ability to stimulate the BH4-dependent
hydroxylation of phenylalanine21.
PCD activity was also shown to be present in human liver, and its absence
is concomitant with the formation of 7-substituted pterins22.
Unexpectedly, the primary structure of PCD is identical with a protein
of the cell nucleus, named dimerization cofactor of hepatocyte nuclear
factor 1a (DCoH), reported to have general transcriptional function23-25.
However, the evolution of the dual function of regulation of PAH activity
and transcription activation in a single protein us unprecedented.
In the last step of BH4 recycling, the q-dihydrobiopterin
is reduced back to BH4 by the NADH-dependent dihydropteridine
reductase (DHPR). There is no evidence that DHPR has regulatory
properties. However, it has been shown that a number of
drugs can inhibit the enzyme's activity, both in vivo and
in vitro. For instance, methotrexate, an antineoplastic
drug commonly used in treatment of cancer, can significantly
inhibit DHPR activity26.

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