Phenylalanine-4-monooxygenase activity

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

FIGURE 8.28 Phenylalanine and tyrosine catabolism. Phenylalanine is converted to tyrosine by phenylalanine monooxygenase. This enzyme requires tetrahydrobiopterin as a cofactor. This cofactor is synthesized in the body from GTP and must be in the fully reduced, tetrahydro form to be active. The cofactor is converted to the dihydro form in the course of the reaction. A separate enzyme, which uses NADPH as a reducing agent, catalyzes the reduction of dihydroprotein back to tetrahydrobiopterin. Oxygen is the cosubstrate of phenylalanine monooxygenase, as well as of two other enzymes, in the pathway shown. 

Individuals with PKU lack phenylalanine hydroxylase (pheny-lalanine-4-monooxygenase) activity so they cannot synthesize tyrosine from phenylalanine. Tyrosine is therefore an essential amino acid for these patients. 

Zaylak M, -J., Curtius H-Ch, Leimbacher W., and Redweik U (1977) Quantitation of deuterated and non-deuterated phenylalanine and tyrosine in human plasma using the selective ion monitoring method with combined gas chromatography-mass spectrometry. Application to the m vivo measurement of phenylalanime-4-monooxygenase activity. / Chromatogr 142, 523-531... 

Francisco, W.A., Blackburn, N.J. and Klinman, J.P. (2003). Oxygen and hydrogen isotope effects in an active site tyrosine to phenylalanine mutant of peptidylglycine alpha-hydroxylating monooxygenase mechanistic implications. Biochemistry 42, 1813-1819... 

Tyrosinase is a monooxygenase which catalyzes the incorporation of one oxygen atom from dioxygen into phenols and further oxidizes the catechols formed to o-quinones (oxidase action). A comparison of spectral (EPR, electronic absorption, CD, and resonance Raman) properties of oxy-tyrosinase and its derivatives with those of oxy-Hc establishes a close similarity of the active site structures in these proteins (26-29). Thus, it seems likely that there is a close relationship between the binding of dioxygen and the ability to "activate" it for reaction and incoiporation into organic substrates. Other important copper monooxygenases which are however of lesser relevance to the model studies discussed below include dopamine p-hydroxylase (16,30) and a recently described copper-dependent phenylalanine hydroxylase (31). 

Tire tetrahydrobiopterin formed in this reaction is similar in structure to a reduced flavin. The mechanism of its interaction with 02 could reasonably be the same as that of 4-hydroxybenzoate hydroxylase. However, phenylalanine hydroxylase, which catalyzes the formation of tyrosine (Eq. 18-45), a dimer of 451-residue subunits, contains one Fe per subunit,113 313i whereas flavin monooxygenases are devoid of iron. Tyrosine hydroxylase416 193 and tryptophan hydroxylase420 have very similar properties. All three enzymes contain regulatory, catalytic, and tetramerization domains as well as a common Fe-binding motif in their active sites.413 421 4213... 

A. The patient, despite being put on a low-Phe diet, exhibits neurologic problems resulting from an inability to synthesize catecholamine and indoleamine neurotransmitters. This is caused by a deficiency in dihydropteridine reductase (DHPR). DHPR regenerates tetrahydro-biopterin (BH ), which is oxidized to dihydrobiopterin by phenylalanine hydroxylase, as well as tyrosine hydroxylase and tryptophan hydroxylase (tryptophan 5-monooxygenase). If phenylalanine hydroxylase were deficient, a diet low in Phe would alleviate the effects. Since the urinary biopterin concentration is elevated, a deficiency in GTP cyclohydrolase I is eliminated because that is an enzyme in the biosynthetic pathway of BH. Phe hydroxylase, Tyr hydroxylase, and Tip hydroxylase activities are low because of a lack of BH. ... 

Fig. 12. Hypothetical high-valent hydroxylating species for different monooxygenases. For the pterin monooxygenases, the N represents histidine residues coordinated to the catalyrtically active Fe in tyrosine, phenylalanine, and tryptophan hydroxylases (.140, 141). The two squares for cyttochrome F450 represent the porphyrin ring, and the + represents a rr-radical delocalized over this ring.

The activity of phenylalanine-4-monooxygenase (EC 1.14 16.1) has been monitored in vivo in man by monitoring the conversion of phenylalanine-ds to tyrosine-d4 (Zaylak et al., 1977)... 

The first step of flavonone biosynthesis begins with the deamination of the amino acid phenylalanine or tyrosine by a phenylalanine ammonia-lyase (PAL) or a tyrosine ammonia-lyase (TAL), which affords cinnamic acid and p-coumaric acid, respectively (Figure 6.36). The formed cinnamic acid is first hydroxylated to p-coumaric acid by a membrane-bound P450 monooxygenase, cinnamate 4-hydroxylase (C4H), and then activated to p-coumaroyl-CoA by a 4-coumarate-CoA ligase (4CL). 4CL catalyzes also the conversion of caffeic acid, feruhc acid, and cinnamic acid to caffeoyl-CoA, feruloyl-CoA, and cinnamoyl-CoA, respectively. 

There is a possibility that a couple of nonheme monooxygenases other than methane monooxygenase possess paired iron centers, but most of the iron proteins are suggested to contain a monomeric iron site. These include tyrosine hydroxylase [13-15] phenylalanine hydroxylase [16, 17], and isopenicillin N synthase [18,19]. Unfortunately, the active site structures of this class of enzymes have not been elucidated to date. Neither have the reaction mechanisms of these understood (recently, the crystal structure of isopenicillin N synthase has been reported) [20]. The function of this enzyme is not hydroxylation reaction but catalyzes the cyclization of L-5-(a-aminoadipoyl)-L-cysteinyl-D-valine to afford isopenicillin, while the catalytic reaction of this enzyme is assumed to include the reductive activation of dioxygen which affords water and high valent 0x0 iron species as... 

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