Phenylalanine, hydroxylation

Figure 9-6. Synthesis of tyrosine from phenylalanine. Hydroxylation of phenylalanine to tyrosine is one of several reactions in the body that require tetrahydrobiopterin as a cofactor to provide electrons and hydrogen as reducing equivalents.

Recently it was discovered that cofactor activity with phenylalanine hydroxylase is not limited to tetrahydropterin derivatives. Thus, the substituted pyrimidines 2,4,5-triamino-6-hydroxypyrimidine (21) and 5-(benzylamino)-2,4-diamino-6-hydroxypyrimidine (22) are active in the L-phenylalanine hydroxylating system (78BBR(85)1614, 79JBC(254)5150, 80JBC(255)7774). The amine substituent at C-5 of (21) and (22) is apparently required for... 

The first step in the liver pathway is catalyzed by phenylalanine hydroxylase. Tetrahydrobiopterin is a cofactor. This redox cofactor is also required for the hydroxylation of tyrosine to form L-dopa (Chapter 16) and for the hydroxylation of tryptophan to form 5-hydroxy tryptophan. The structure of tetrahydrobiopterin is given in Figure 20.23. In the process of phenylalanine hydroxylation, the tetrahydrobiopterin is oxidized to dihydrobiopterin. The reduced form is then recovered via NADH and dihydrobiopterin reductase, as shown in Figure 20.23. Dihydrobiopterin, although similar in structure to folic acid, is synthesized in the human organism from GTP. 

A schematic representation of the phenylalanine hydroxylation reaction is shown in Figure 19-2. PAH reduces and cleaves molecular oxygen into two hydroxyl groups. One of the hydroxyl functional groups is found in the reaction product tyrosine, while the other oxygen atom is used to modify the reaction cofactor BII4, creating the pterin 4a-carbinolamine. 

Ionizing-radiation tissue injury Fission of H2O molecule to 011 and IP DNA damage Decrease in tissue —SH pool tyrosine, phenylalanine hydroxylation... 

This enzyme also catalyzes conversion of dihydrofolate (FH2) to tetrahydrofolate (FH4), and folic acid contains a pteridine ring system (see the discussion of one-carbon metabolism in Chapter 27). However, regeneration of tetrahydrobiopterin by the dihydrofolate reductase reaction, however, is too slow to support normal rates of phenylalanine hydroxylation. 

The provision of reducing equivalents to phenylalanine hydroxylase is dependent on reduction of dihydrobiopterin by NADH catalyzed by the enzyme dihydropteridine reductase, as shown in Figure 38-2. This reduction is dependent on the availability of biopterin and therefore on the biopterin synthetic pathway. Thus any genetic or protein folding defect in either dihydropteridine reductase or the biopterin biosynthetic enzymes would compromise the efficacy of phenylalanine hydroxylation to tyrosine resulting in hyperphenylalaninemia and also phenylketonuria resulting from inaease transamination of phenylalanine to phenylpyruvate. 

Regeneration of BH4 is an essential part of the phenylalanine hydroxylating system (see also Cofactor functions ). During the catalytic event of aromatic amino acid hydroxylases, molecular oxygen is transferred to the corresponding amino acid and BH4 is oxidized to BH4-4a-carbinolamine (Figure 15). " °° Two enzymes are... 

Kaufman and his associates [73] reinvestigated the role of the two enzyme fractions in phenylalanine hydroxylation. The reaction was carried out in vitro in the presence of extensively purified rat and sheep liver enzymes. Two cofactors were necessary for the overall reaction a pteridine and NADPH. Dihydropteridine [74, 75] has been established as the cofactor of phenylalanine hydroxylase (see Fig. 3-21). 

Kaufman, S. Phenylalanine hydroxylation cofactor in phenylketonuria. Science 128, 1506-1508 (1958)... 

Kaufman, S. The structure of the phenylalanine-hydroxylation cofactor. Proc. nat. Acad. Sci. (Wash.) 50, 1085-1093 (1963)... 

Coumaric acid 5.24) is the product derived by loss of ammonia from tyrosine (5.75). Much more commonly this acid derives by hyd-roxylation of cinnamic acid 5.23) formed by ammonia loss from phenylalanine hydroxylation is accompanied by the usual NIH shift (Section 1.3.2) for this para-hydvoxyXdXion (also or/Ao-hydroxylation [14]). Up to two further phenolic hydroxy-groups may be introduced on 5.24), Scheme 5.4 [15, 16]. It is interesting to note that in... 

Conversion of N-benzoyl-L-phenylalanine into L-phenylalanine Hydroxylation... 

Phenylalanine hydroxylase is like p-hydroxyphenylpyruvate oxidase (see below) in its requirement for two enzyme components. It is particularly interesting that one of these components (Fraction II) can be replaced by acetone powder extracts of liver from rabbit, calf, dog, and pig, and by acetone powder extracts of kidney and heart from calf and hog, although these oi ans contain no phenylalanine hydroxylase activity. Fraction II appears to be wide distributed, and to have fimctions other than these connected with phenylalanine hydroxylation. Although two enzyme components are involved in the system, no evidence for an intermediate between phenylalanine and tyrosine has been found (557), nor has it been possible to separate the process into two steps. 

From evidence at hand, therefore. Fraction I is specifically involved in phenylalanine hydroxylation and may form oxygen complexes. Fraction II is a widely distributed ferroprotein, probably a DPNH oxidase which can utilize oxygen itself as an electron acceptor... 

In phenylketonuria (typical or classical variety) the phenylalanine hydroxylating system is inactive, all the evidence pointing to lack of phenylalanine hydroxylase [45]. As a consequence, phenylalanine accumulates in the tissues and body fluids, reaching concentrations as high as 120 mg per 100 ml plasma (normal 0.9). The concentration of tyrosine is about... 

Kaufman, S., 1971, The phenylalanine hydroxylating system from mammalian liver, Adv. Enzymol. 35 245. 

In phosphate buffer, pH 6.8, the conversion of the intermediate to the 7,8-dihydropteridine is so rapid that in 1 minute the spectrum is similar to the one shown in Tris buffer after 90 minutes. The compound after this brief exposure to phosphate is inactive both in the TPNH oxidation assay and in the phenylalanine hydroxylation system. 

A solution of the intermediate prepared by the dye-oxidation procedure was tested in the phenylalanine hydroxylating system. The results, shown in Table III, demonstrated that the compound was active only in the... 

Activity of Oxidized Pteridine Intermediate in Phenylalanine Hydroxylation System ... 

It has already been mentioned that, when tetrahydropteridines are used in the phenylalanine hydroxylation system in place of the rat liver... 

The activity in the phenylalanine hydroxylation system of the product of the reaction between TPNH and the intermediate strongly suggests that this product is the tetrahydropteridine. More direct evidence in support of this idea was obtained by separating this product from the TPNH on a Dowex-l-Cl column and examining its spectrum. It was found that the initial spectrum corresponded to that of a mixture of 85% tetrahydro- and 15% 7,8-dihydropteridine. The spectrum of the sample was redetermined after various periods and the changes were indistinguishable from those reported for authentic tetrahydropteridine when stored in 0.1 M phosphate, pH 6.8. 

To decide between the remaining two possibilities an experiment was performed b ed on some recent observations concerning the optical specificity of tetrahydrofolate in the phenylalanine hydroxylating reaction. It has been found that only one isomer of tetrahydrofolate is active in the hydroxylating system the isomer which is synthesized enzymically in either the dihydrofolic reductase (Osborn and Huennekens, 1958) or the glutamic transformylase system (Silverman et al., 1957) is almost completely inactive (Kaufman, unpublished). 

The /-L-tetrahydrofolate used in Experiments 5, 6, and 7 was generated enzymically from 5-formyltetrahydrofolate by anaerobic incubation in the presence of the glutamate transformylating system. A total of 0.045 nmole of /-L-tetrahydrofolate was liberated. To all tubes which were to be preincubated (2, 3, 5, 6, and 7), 0.5 nmole of TPNH as added, and the samples were then incubated for 2i hours aerobically or anaerobically as indicated in the table. Finally, a sample was assayed in the phenylalanine-hydroxylating system. 

G. Behavior of Phenylalanine Hydroxylation Cofactor and Tetrahyd ropteridi nes... 

From the results of studies on the phenylalanine-hydroxylation system, as well as from those on other hydroxylation reactions, it was anticipated that a reducing agent might be required for the conversion of dopamine to norepinephrine. It was found that ascorbate could stimulate the reaction even in adrenal particles and the requirement for ascorbate became more complete as the enzyme was purified. With the purified enzyme, the for ascorbate was found to be about 6 X 10 M. The specificity of the ascorbate requirement is shown in Table VII. Of the... 

As might be expected from this formulation, one can demonstrate a dopamine-dependent oxidation of ascorbate. This reaction can be followed by measuring the ascorbate disappearance spectrophoto-metrically or by utilizing the 2,6-dichlorophenolindophenol titration method. Figure 15 shows the spectrophotometric demonstration of the dopamine stimulation of ascorbate disappearance. These results, as well as those of the balance studies, are similar to those obtained with the phenylalanine-hydroxylating system and support the idea that the fundamental mechanism is the same in both cases. Besides the utilization of different electron donors, a difference which is probably trivial in... 

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