Tyrosine hydroxylase (TyrH) catalyzes the pterin-dependent hydroxylation of
tyrosine to form dihydroxyphenylalanine. The enzyme requires one atom of ferrous iron
for activity. Using deuterated 4-methylphenylalanine substrates, intrinsic primary and
secondary isotope effects of 9.6 ± 0.9 and 1.21 ± 0.08 have been determined for benzylic
hydroxylation catalyzed by TyrH. The large, normal secondary isotope effect is
consistent with a mechanism involving hydrogen atom abstraction to generate a radical
intermediate. The similarity of the isotope effects to those measured for benzylic
hydroxylation catalyzed by cytochrome P-450 suggests that a high-valent, ferryl-oxo
species is the hydroxylating species in TyrH. Uncoupled mutant forms of TyrH have
been utilized to unmask isotope effects on steps in the aromatic hydroxylation pathway
which also implicate a ferryl-oxo intermediate. Inverse secondary isotope effects were
seen when 3,5-2H2-tyrosine was used as a substrate for several mutant enzyme forms.
This result is consistent with a direct attack by a ferryl-oxo species on the aromatic ring
of tyrosine forming a cationic intermediate. Rapid-freeze quench Mössbauer studies have provided preliminary spectroscopic evidence for an Fe(IV) intermediate in the reaction
catalyzed by TyrH.
The role of the iron atom in the regulatory mechanism has also been investigated.
The iron atom in TyrH, as isolated, is in the ferric form and must be reduced for activity.
The iron can be reduced by a number of one-electron reductants including
tetrahydrobiopterin, ascorbate, and glutathione; however, it appears that BH4 (kred = 2.8 ±
0.1 mM-1 s-1) is the most likely candidate for reducing the enzyme in vivo. A one-electron
transfer would require a pterin radical. Rapid-freeze quench EPR experiments aimed at
detecting the intermediate were unsuccessful, suggesting that it decays very rapidly by
reducing another equivalent of enzyme. The active Fe(II) form can also become oxidized
by oxygen (210 ± 30 M-1 s-1); this increases the affinity of catecholamine inhibitors.
Serine 40 can be phosphorylated to relieve the inhibition; however, results with S40E
TyrH show phosphorylation does not have an effect on the rate constant for reduction of
the enzyme but causes a 40% decrease in the rate constant of oxidation.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/3817 |
| Date | 16 August 2006 |
| Creators | Frantom, Patrick Allen |
| Contributors | Fitzpatrick, Paul F. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 1187940 bytes, electronic, application/pdf, born digital |
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