The pterin-dependent amino acid hydroxylases (AAH) are a group of mononuclear, nonheme monooxygenases that catalyze the oxidation of aromatic amino acids necessary for the bio- synthesis of a variety of neurotransmitters.74,75 The three primary enzymes that have been
RHN SH
NH H
COOH O
FeII(IPNS), O2
RHN S
NH H
COOH O
FeII O2
RHN S
NH H
COOH O
FeII (O2)2-
RHN S
N H
COOH O
FeIV
H2O O
RHN S
N HOOC O
H2O, FeII(IPNS)
Figure17 Proposed mechanism for isopenicillin N synthase.
isolated and characterized are phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TYRH) and tryptophan hydroxylase (TRPH). Other oxygenases of this class, anthranilate hydroxylase,76 mandelate hydroxylase77and glyceryl-ether monooxygenase,78have been identified but have not been isolated or characterized in detail.60 PAH, TYRH and TRPH utilize the 2e/2Hþreduc- tant tetrahydrobiopterin (BH4:(6(R)-L-erythro-5,6,7,8-tetrahydropterin) as cofactor, Figure 20.60 PAH is the AAH that has been the most widely studied owingto its availability and
Fe2+
N
N N
N O
O O
Ph
[FeII(L)(BF)]+
Fe2+
N
N N
N O
O O
Ph
Fe3+
N
N N
N O
O O
Ph O2
Fe3+
N
N N
N O
O O
Ph O O Fe2+
N
N N
N O
O Ph O O C O
Fe4+
N
N N
N O
O O Ph
Fe2+
N
N N
N O
O Ph S
SO
[FeII(L)(OBz)]+
Autoxidation Fe3+
N
N N
N O
[FeIII2(O)(L)2(OBz)2]2+ Ph O
O Fe3+
O O PH
N
N N
N
Figure18 Proposed mechanism for the oxygenation of benozoylformateiron(II) complexes.
O O +
OH O
+
no reaction
Figure19 Olefin oxidation reactions of [Fe(BF)(TpMe2)(CH3CN)].
stability. PAH is responsible for maintainingphysiological L-Phe homeostasis. A disfunction of this enzyme results in the disease known as phenylketonuria (PKU), which if left untreated leads to severe mental retardation and premature death.74 This disease represents one of the most common metabolic disorders of clinical significance in children. TYRH is the enzyme respon- sible for the formation of dihydroxyphenylalanine (L-dopa) whose physiological concentrations have been linked to disorders such as Parkinson’s disease.74 L-dopa is then converted to other neurotransmitters such as norepinephrine and epinephrine. Finally, TRPH converts the trypto- phan amino acid to 5-hydroxy-tryptophan, which is the first step in the biosynthesis of the neurotransmitter serotonin.74 Each of these proteins share a notable similarities in their sequences: 65% sequence identity and 80% sequence homology in the catalytic domains which contain the nonheme iron center.75
8.14.4.2 ActiveSites for theAmino Acid Hydroxylases
Upon expression and purification, the proteins are retrieved in the inactive, ferric state due to the ease of oxidation of the ferrous center duringisolation. It has been shown that the cofactor, BH4, can act as a reductant for the ferric ion and as such, serves to maintain the protein in its active ferrous state for the enzymes PAH and TYRH.79,80 Recent spectroscopic studies have shown the iron center in PAH to be coordinated in a distorted, octahedral geometry.81,82 The catalytic domain has an / fold that makes a basket–like arrangement of helices and loops.0 The active site is situated10 A˚ away from the protein surface and is coordinated through three endogenous protein ligands: His285,His290and Glu330,Figure 21a, thereby conservingthe 2-His-1-carboxylate facial triad bindingmotif of mononuclear, nonheme oxygenases.1 The crystal structure for tyrosine hydroxylase shows a square pyramidal arrangement wherein the 2-His-1-carboxylate motif occurs via coordination through the three endogenous protein ligands His331, His335 and Glu375, with two water molecules occupyingthe remainingcoordination sites,Figure 21b.75This five-coordinate structure has recently been debated based on spectroscopic evidence for a six coordinate iron center83 and current studies are attemptingto resolve/explain the discrepancies between the crystallographic and spectroscopic data for the protein.
The AAH enzymes show remarkable specificity for their respective substrates; a characteristic governed by their regulatory domains. Each active site is situated within a hydrophobic pocket with negative electrostatic potential at the active site75offeringa suitable bindingenvironment for
NH H N
NH N
R
O
NH2 + O2
NH HN
NH N
R
O
NH2
OH BH4
4a-hydroxy-BH4
OH
Phenylalanine hydroxylase
Tyrosine hydroxylase
OH OH
HO
Tryptophan hydroxylase HN
HN OH
Figure20 Transformation carried out by pterin-dependent hydroxylases.
amphipatic molecules such as substrates, the pterin cofactor or inhibitors. A major focus in the studies of these proteins involves elucidatingthe details surroundingthe specific interactions between the substrate, cofactor and iron center.
8.14.4.3 Mechanism for Substrate Hydroxylation
The currently accepted mechanism by which substrate hydroxylation occurs in PAH is depicted in Figure 22.74MCD and XAS studies of the restingstate inactive ferric and active ferrous enzymes indicate the presence of a slightly distorted six-coordinate iron site, a conclusion in agreement
FeIII H2O
O Glu330 O
N NH
OH2
N NH HOSer344
His285
His290 H2O
H2O Tyr325
O
O Glu285
OH2
(a)
FeIII N
NH His331 O N H2O
OH2 NH
His335 Glu376 O
(b) Figure21 Iron coordination environments for PAH and TYRH.
NH HN
NH N
R
O NH2
O2 Fe(II)
NH HN
NH N
R
O t-Bu
O2 Fe(II)
CH2 C H NH3+
COO2– C
H2 C H NH3+
COO2–
NH HN
N N
R
O NH2
O O
Fe(II)
CH2 C H NH3+
COO2–
NH HN
N N
R
O NH2
O O Fe(II)
CH2 C H NH3+
COO2–
NH HN
N N
R
O NH2
OH
CH2C H NH3+
COO2– Fe(IV)O NH
HN
N N
R
O NH2
OH
CH2C H NH3+
COO2– FeO
H H NH
HN
N N
R
O NH2
H OH
CH2C H NH3+
COO2– Fe(II)
HO
+
-
Figure22 Proposed mechanism for phenylalanine hydroxylation by PAH.
with crystallographic studies of various truncated forms of oxidized PAH. This coordination environment is maintained in the presence of either cofactor alone or substrate alone.84,85 However, prior to enzymatic turnover, PAH undergoes an allosteric activation induced by the bindingof Phe to a site distinct from the catalytic site. Interestingly, once PAH is activated, the ferrous site adopts a square-pyramidal geometry caused by the loss of a single water ligand.86It is this form of the ferrous enzyme that is catalytically active. The proposed mechanism indicates that the initial reaction involves the interaction of dioxygen with the reduced pterin cofactor to afford the C4a-hydroperoxy-H2biopterin species, a step that activates the kinetically inert OO bond of dioxygen by converting it to an alkyl hydroperoxide moiety. It is important to note that there is no experimental evidence suggesting that either the reduced cofactor or the substrate molecule coordinates to the ferrous center in the active site. The next step is thought to involve the coordination of the alkylperoxide to the ferrous iron center88followed by subsequent heterolytic cleavage of the OO bond to generate a high valent iron-oxo species and C4a-hydroxyl-H2bio- pterin, a form of the cofactor observed duringcatalytic turnover. The analogy has been made between this iron oxo species and the one that has been postulated for heme systems such as cyt- P450.89 Attack of the electrophilic ferryl species on the phenylalanine substrate then forms a cationic substrate intermediate90 that, upon cleavage of the iron species, regenerates the ferrous ion leavingthe hydroxylated product tyrosine.74,91The oxidation of Phe to Tyr is known to occur via a pathway that induces an NIH shift of para substituents on Phe (2H,3H, Cl, CH3), a process consistent with an oxygen atom transfer step. Interestingly, insights into the mechanism of wildtype PAH have allowed for a deeper understandingof the chemical basis of phenylketonuria as parallel spectroscopic and enzyme mechanistic studies on selected missense mutants that give rise to PKU in the human population have been reported. Such studies will enable the identifica- tion of the chemical basis of PKU.92
To date, no functional model systems have been developed to probe the reactivity and structural characteristics of the family of aromatic nonheme iron tetrahydropterin-dependent monooxygenases. Currently, all mechanistic information regarding the reaction pathways involved in PAH has been derived from in vitro studies on the protein. For a more detailed description of the evidence, the reader is directed to the accompanyingreferences cited above.