Phenylalanine hydroxylase, properties

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). 

The liver is also the principal metabolic center for hydrophobic amino acids, and hence changes in plasma concentrations or metabolism of these molecules is a good measure of the functional capacity of the liver. Two of the commonly used aromatic amino acids are phenylalanine and tyrosine, which are primarily metabolized by cytosolic enzymes in the liver [1,114-117]. Hydroxylation of phenylalanine to tyrosine by phenylalanine hydroxylase is very efficient by the liver first pass effect. In normal functioning liver, conversion of tyrosine to 4-hy-droxyphenylpyruvate by tyrosine transaminase and subsequent biotransformation to homogentisic acidby 4-hydroxyphenylpyruvic acid dioxygenase liberates CO2 from the C-1 position of the parent amino acid (Fig. 5) [1,118]. Thus, the C-1 position of phenylalanine or tyrosine is typically labeled with and the expired C02 is proportional to the metabolic activity of liver cytosolic enzymes, which corresponds to functional hepatic reserve. Oral or intravenous administration of the amino acids is possible [115]. This method is amenable to the continuous hepatic function measurement approach by monitoring changes in the spectral properties of tyrosine pre- and post-administration of the marker. 

The following is review on the molecular and physical properties of this class of monooxygenases, which are also known as hydroxylases. A typical monooxygenase reaction is the hydroxylation of an alkane to an alcohol which involves a reduced cosubstrate that reduces a second atom within the O2 molecule to form water. Flavin-containing monooxygenases include lysine oxygenase and 4-hydroxybenzoate hydroxylase. Reduced pteri-dines are involved in the phenylalanine hydroxylase and tryptophan hydroxylase reactions. See also Cytochrome P-450... 

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... 

The general scheme of the biosynthesis of catecholamines was first postulated in 1939 (29) and finally confirmed in 1964 (Fig. 2) (30). Although not shown in Figure 2, in some cases the amino acid phenylalanine [63-91-2] can serve as a precursor it is converted in the liver to (-)-tyrosine [60-18-4] by the enzyme phenylalanine hydroxylase. Four enzymes are involved in E formation in the adrenal medulla and certain neurons in the brain tyrosine hydroxylase, dopa decarboxylase (also referred to as L-aromatic amino acid decarboxylase), dopamine-P-hydroxylase, and phenylethanolamine iV-methyltransferase. Neurons that form DA as their transmitter lack the last two of these enzymes, and sympathetic neurons and other neurons in the central nervous system that form NE as a transmitter do not contain phenylethanolamine N-methyl-transferase. The component enzymes and their properties involved in the formation of catecholamines have been purified to homogeneity and their properties examined. The human genes for tyrosine hydroxylase, dopamine- 3-oxidase and dopa decarboxylase, have been cloned (31,32). It is anticipated that further studies on the molecular structure and expression of these enzymes should yield interesting information about their regulation and function. 

Abita, J.P., Parniak, M., and Kaufman, S., The activation of rat liver phenylalanine hydroxylase by limited proteolysis, lysolecithin, and tocopherol phosphate. Changes in conformation and catalytic properties, J. Biol. Chem. 259 (23), 14560-14566,1984. 

The enzyme has been purified from rat and human livers. The properties of the human enzyme are essentially similar to those of the rat enzyme [204, 205]. Phenylalanine hydroxylase has also been purified from rat kidney. Available methods of investigation suggest that the kidney enzyme is similar to that of liver [206]. The human liver enzyme exists in a soluble and particulate form. The particulate and soluble enzymes have identical pH optima and K s apparent for phenylalanine and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine [207]. 

In contrast to true oxygenases, most hydroxylases are rather unstable and have not been prepared in a highly purified form. The properties of phenylalanine hydroxylase will be described elsewhere in this treatise by Kaufman (Chapter 4). 

Kynurenine Hydroxylase. Kynurenine is hydroxylated by an enzyme prepared from mitochondria of animal livers. The properties of this enzyme are very similar to those described from the hydroxylation of other aromatic amines, steroids, and phenylalanine molecular oxygen is consumed and an equivalent of TPNH is oxidized simultaneously with kynurenine oxidation (VIII). 

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