A specific kinetic assay for phenylalanine hydroxylase

Ayling, June E.; Pirson, R.; Pirson, Wolfgang; Boehm, Günther

doi:10.1016/0003-2697(73)90454-5

Cited by 47 publications

(34 citation statements)

References 5 publications

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“…The acid-base catalyzed rearrangement 214 of quinonoid 6-methyl-7,8-dihydro(6H)pterin (q-6-MePH 2 , oxidized cofactor) to 6-methyl-7,8-dihydropterin (6-MePH 2 , nonreducible to 6-MePH 4 in this system) was considerably slower (t 0.5 23 min at pH 7.2, 30ЊC) 215 than the rearrangement of quinonoid 6,7-dimethyl-7,8-dihydro(6H)pterin (q-6,7-Me 2 PH 2 ) to 6,7-dimethyl-7,8-dihydropterin (6,7-Me 2 PH 2 , t 0.5 11.8 min at pH 7.6, 25ЊC) 214 and produced lower concentrations of recyclable cofactor in the 6,7-dimethylpterin. Also tetrahydropterins readily undergo autoxidation in aqueous buffer 214,216 (the solubility of oxygen under 1 atmosphere of air in aqueous buffer is 0.25 mM) 217 with the formation of quinonoid species. Since catalase, which is known to inhibit the autoxidation of tetrahydropterins [217][218][219] probably by specific removal of H 2 O 2 (product of the oxidation), 219 was not included in this assay then the rates of NADH oxidation were not exact measures of the monooxygenase reaction (although they were probably close to them, as initial rates were measured).…”

Section: Enzyme Assaysmentioning

confidence: 99%

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

Taguchi¹,

Armarego²

1998

Med. Res. Rev.

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Section: Enzyme Assaysmentioning

confidence: 99%

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

Taguchi¹,

Armarego²

1998

Med. Res. Rev.

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“…Phenylalanine hydroxylase was assayed at 25 O C unless stated otherwise by either direct measurement of the rate of formation of oxidized cofactor (Ayling et al, 1973) (assay l), measurement of the rate of tyrosine formation by following the change in absorbance at 275 nm (Shiman et al, 1979) (assay 2), or measurement of tyraine formation from the fluorescence of the nitrosonaphthol derivative (Waalkes & Udenfriend, 1957) …”

Section: -2960/84/0423-1295%01 So10mentioning

confidence: 99%

Reductive activation of phenylalanine hydroxylase and its effect on the redox state of the non-heme iron

Wallick¹,

Bloom²,

Gaffney

et al. 1984

Biochemistry

118

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Phenylalanine hydroxylase undergoes an obligatory prereduction step in order to become catalytically active as shown by stopped-flow kinetics and by measuring tyrosine formation at limiting 6-methyltetrahydropterin levels. This initial step requires oxygen and involves conversion of 6-methyltetrahydropterin directly to the quinonoid form with or without phenylalanine. The EPR spectrum of the resting enzyme (geff = 9.4-8.7, 4.3 and geff = 6.7, 5.4) is consistent with two species possessing distinctively different ligand environments for the non-heme, high-spin Fe3+. The intensity of the geff N 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the geff E 6.7-5.4 region correlates with the activity of the enzyme. The I n our attempts to understand the mechanism of action of phenylalanine hydroxylase (PAH) from rat liver, we (Gottschall et al., 1982) recently confirmed and extended an earlier observation by Fisher et al. (1972) that a tightly bound non-heme iron was necessary for the activity of the enzyme. We demonstrated that (1) the correct stoichiometry is one atom per subunit, (2) the enzymatic activity is proportional to the iron content, and (3) the iron can be removed from the enzyme and restored with nearly complete recovery of initial activity. In this paper we corroborate and expand the independent discovery (Marota & Shiman, 1984) that PAH is initially reduced to the catalytically active enzyme by the concomitant oxidation of 6-methyltetrahydropterin (6MPH4) directly to the quinonoid form in a stoichiometric reaction requiring oxygen but without the formation of reduced forms of oxygen such as superoxide and hydrogen peroxide, or of tyrosine. In this paper we (1) propose a two-step kinetic sequence for PAH activation and its catalytic turnover based on stopped-flow kinetic and complimentary product data, (2) define by EPR that the resting state of the activatable enzyme is associated with signals observed at geff = 6.7 and 5.4 consistent with high-spin Fe3+ (S = (3) show that the additional EPR signal observed at geff = 4.3 is associated with a form of the enzyme that is incapable of turnover, and (4) link the prereduction step to the conversion of PAH from an Fe3+ to an Fez+ state. Finally, we demonstrate that dithionite can substitute for 6MPH4 in the prereduction step and that the addition of one electron/subunit is sufficient to impart tightly coupled turnover. Experimental Procedures MaterialsDoubly distilled deionized water was used throughout. All reagents were of the highest grade commercially available. 0006-2960/84/0423-1295%01 SO10latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of ophenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fez+ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing ...

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“…H4 cells were grown to confluency in 75-cm2 flasks (Lux Scientific, Thousand Oaks, CA) in modified Swim's medium S-77 supplemented with 5% (vol/vol) fetal bovine and 10% horse sera (1,2). The harvesting and extraction of postconfluent cultures for assay of phenylalanine hydroxylase by the method of Ayling et al (6) have been described (1)(2)(3). One enzyme unit is defined as the phenylalanine-dependent oxidation of 1 nmol of 6,7-dimethyltetrahydropterin to 6,7-dimethyldihydropterin or the formation of 1 nmol of tyrosine per min at 27°C.…”

mentioning

confidence: 99%

Translation of phenylalanine hydroxylase-specific mRNA in vitro: evidence for pretranslational control by glucocorticoids.

Chiappelli¹,

Haggerty²,

Lynch³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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We have found that the induction of phenylalanine hydroxylase by hydrocortisone and serum in confluent cultures of H4--E-C3 rat hepatoma cells is accompanied by an increase in polysomal mRNA specific for phenylalanine hydroxylase, as measured by translation in a cell-free protein-synthesizing system. Thus, the induction is mediated largely, if not entirely, by a pretranslational mechanism, possibly by stimulation of the transcription of the phenylalanine hydroxylase gene.It has been shown that two cultured rat hepatoma cell lines, H4-II-E-C3 (H4) and MH1C1, contain phenylalanine hydroxylase but at a very low level (1). Addition of hydrocortisone or dexamethasone at submicromolar concentrations and of steroid-free blood serum, from diverse mammalian species, to confluent cultures of the H4 cells caused an increase in the hydroxylase content of the cells to levels comparable with those found in normal rat liver (1, 2). This increase resulted from an increase in the amount of hydroxylase protein and not from activation of an inactive (pro)enzyme (3). The stimulation by glucocorticoids was shown to be mediated by an increase in the rate of the synthesis of the enzyme (4) without any change in the rate of its degradation (5).By the use ofa cell-free protein-synthesizing system ofwheat germ and specific antiserum against rat liver phenylalanine hydroxylase, we now show that the induction of the hydroxylase by hydrocortisone and serum in H4 cells is associated with an increase of polysomal mRNA specific for phenylalanine hydroxylase (mRNAph).METHODS AND MATERIALS Cell Cultures and Assay for Phenylalanine Hydroxylase. H4 cells were grown to confluency in 75-cm2 flasks (Lux Scientific, Thousand Oaks, CA) in modified Swim's medium S-77 supplemented with 5% (vol/vol) fetal bovine and 10% horse sera (1, 2). The harvesting and extraction of postconfluent cultures for assay of phenylalanine hydroxylase by the method of Ayling et al. (6) have been described (1-3). One enzyme unit is defined as the phenylalanine-dependent oxidation of 1 nmol of 6,7-dimethyltetrahydropterin to 6,7-dimethyldihydropterin or the formation of 1 nmol of tyrosine per min at 27°C. Protein concentrations were measured by dye-binding (7).To prepare the basal and fully induced cells used for the preparation of poly(A)+RNA (see Table 1 and Fig. 2), postconfluent cultures of H4 cells were first washed with modified S-77 medium and then given either the same medium without serum and hydrocortisone or serum-containing medium supple- The incorporation of 14C into total soluble cellular protein was 0.35 ,Ci per flask; ofthis, '3% could be precipitated by antiserum against rat liver phenylalanine hydroxylase.Isolation of Total Polysomal Poly(A)+RNA. Poly(A)+RNA was isolated from disrupted H4 cells by a combination of previously described methods (8-10) with minor modifications, such as the use ofHepes for all buffers. A total of 12 preparations was made; each was derived from cells pooled from sets of 10, 25, 30, or 32 similarly grown cultures. Nine prep...

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A specific kinetic assay for phenylalanine hydroxylase

Cited by 47 publications

References 5 publications

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

Reductive activation of phenylalanine hydroxylase and its effect on the redox state of the non-heme iron

Translation of phenylalanine hydroxylase-specific mRNA in vitro: evidence for pretranslational control by glucocorticoids.

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