Rat liver phenylalanine hydroxylase

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. 

Fisher, D.B., Kirkwood, R., Kaufman, S. Rat liver phenylalanine hydroxylase, an iron enzyme. J. biol. Chem. 247, 5161-5167 (1972)... 

Phenylalanine hydroxylase from rat liver contains a tightly bound non-heme iron that is essential for the activity of the enzyme. The enzyme functions aerobically to produce tyrosine from phenylalanine and a tetrahydropterin.828... 

Robson KJH, Chandra T, MacGillivray TRA, Woo SLC. Polysome immuno-precipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA. Proc Natl Acad Sci USA 1982 79 4701-4705. 

Figure 1. Hydroxylation of 4-suhstituted phenylalanines with phenylalanine hydroxylase from either rat liver or Pseudomonas (DMPH, = dimethyltetra-...

Phenylketonuria can be induced experimentally using various phenylalanine derivatives. Among these are phenylalanine alkylating agents [236] and several o-dihydroxy derivatives [237-239] particularly esculetin (6,7-dihydroxy-coumarin) derivatives were found to be very potent in inhibiting phenylalanine hydroxylase both in vivo and in vitro [240, 241]. This observation led to further structure-activity studies applying different 3- and 4-substituted 6,7-dihydroxy-coumarins. In vitro 4-methyl-, 4-butyl- and 4-pheny -6,7-dihydroxycoumarin, and in vivo esculin, 4-methyl- and 4-phenyl-6,7-dihydroxycoumarin appear to be the most powerful inhibitors of the rat liver enzyme (Table 3,7). 

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). 

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]. 

Ayling, J.E., Pirson, W.D., Al-Janabi, J.M., Helfand, G.D. Kidney phenylalanine hydroxylase from man and rat. Comparison with the liver enzyme. Biochemistry 13, 78-85 (1974)... 

In evaluating food requirements in infancy, one must consider that the infant s enzymic arsenal is not equivalent to that of the adult. We have already mentioned that in guinea pigs and rats, glucose-6-phospha-tase is practically nonexistent until shortly before term, but after term it rises rapidly. In humans, phenylalanine hydroxylase is absent from liver until several weeks after term. If the formation of the enzyme molecules is completely inhibited because of a genetic defect, an inborn error of metabolism develops. In rats, tyrosine transaminase increases immediately after birth and reaches a maximum 12 hours after birth. 

Fig. 2. Phenylalanine/tyrosine ratios and phenylalanine hydroxylase activities in the liver of rats treated with -CPA and/or esculin. (Mean values SEM of four to six experiments.) p < 0.05.

Of the two fractions into which the phenylalanine hydroxylase is separable, I is salted out between 0-40% (NH4)2S04 saturation and II, at 40-80 % saturation. Rat liver is rich in I and it has been purified from this source about seventy- to a hundredfold by Kaufman and Levenberg 188, 189). Fraction II is rich in rabbit liver which, conversely, is low in I. This enzyme has also been purified to about the same degree as I by variations of the procedures used in the purification of enzyme I. 

The search for a specific hydroxylating enzyme was, till recently, fruitless because of technical difficulties. In addition, many early reports were later recognized as misleading. The finding, for instance, that a soluble rat-liver fraction could hydroxylate L-tryptophan at very high concentrations was confirmed. However, this was clearly demonstrated to be due to the presence of phenylalanine hydroxylase (EC. 1.14.3.1), a well-known enzyme which uses tetrahydrobiopterin, a reduced pteridine derivative, as a cofactor. Since the affinity of phenylalanine hydroxylase for tryptophan is very poor and since phenylketonuric patients who totally lack this enzyme seem to be able to synthesize normal amounts of 5-hydroxyindoles, it was concluded that this enzymatic system has no physiological relevance whatsoever in the biosynthesis of S-HT. 

Phenylalanine hydroxylase occurs only in mammalian liver (that is, in the rat, guinea-pig, rabbit, d<, chicken, and human) (see also 259). No activity has been observed in (rat) lung, kidney, brain, or muscle. The system is quite speciOc for L-phenylalanine. Tjrro-sine is not formed from n-phenylalanine, nor are the corresponding p-phenols formed from N-acetyl- or N-chloroacetyl-L-phenylalanine, L-phenylalanine ethyl ester, DL-phenylglycine, phenylserine, phenylpyruvic acid, phenylethylamine, benzoic acid, hippuric acid, cinnamic acid, or mandelic acid (768), or from aniline, acetanilide, tryptophan, kynurenine, anthranilic acid, or phenylacetate (557). This specificity is a distinguishing character of the enzyme, which occurs in the same tissue as the nonspecific aromatic hydroxylase described above. 

A more interesting interaction between an enzyme and phospholipids, because of the complexity of the kinetics, is seen with phenylalanine hydroxylase from rat liver. With its naturally occurring substrates, phenylalanine and tetrahydrobiopterin, plots of v vs. [phenylalanine] are sigmoidal (Kaufman, 1971) propanol converts... 

The results of this experiment suggest that the intermediate in the nonenzymic aerobic oxidation of tetrahydropteridines, as well as in the enzymic conversion of phenylalanine to tyrosine, is the 5,6-dihydro-pteridine or a compound in equilibrium with the S,6-dihydropteridine. The scheme in Fig. 12 shows the pteridine transformations which occur during the hydroxylation reaction. It can be seen that the rat liver enzyme actually catalyzes a coupled oxidation phenylalanine is oxidized to tyrosine while the tetrahydropteridine is oxidized to the dihydropteridine. Phenylalanine hydroxylase, a name which has been used to describe the whole system, should properly be restricted to the rat liver enzyme. The sheep liver enzyme catalyzes a TPNH-mediated reduction of the 5,6-dihy-dropteridine to the 5,6,7,8-tetrahydropteridine and may therefore be called dihydropteridine reductase. For purposes of continuity, however, the enzymes will be referred to as the rat and sheep liver enzymes during this discussion. 

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