Pterin-Requiring Aromatic Amino Acid Hydroxylases

Kaufman, Seymour; Fisher, Daniel B.

doi:10.1016/b978-0-12-333640-8.50013-7

Cited by 119 publications

(110 citation statements)

References 141 publications

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“…Coexistence of these appeared to be necessary for maximal stabilization, since the extent of stabilization was lowered by the omission of any of these. Similar stabilization by a non-ionic detergent, EDTA and glycerol has been observed in the cases of phenylalanine 4-monooxygenase [32] and tryptophan 5-monooxygenase [33], both of which, together with tyrosine 3-monooxygenase, were regarded as a family of enzymes [34]. Although a sulfhydryl agent was effective in restoring the enzyme activity [35], it was ineffective in stabilizing the enzyme.…”

Section: Discussionmentioning

confidence: 93%

Purification and Some Properties of Tyrosine 3‐Monooxygenase from Rat Adrenal

Okuno

Fujisawa

1982

European Journal of Biochemistry

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Tyrosine 3-monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1600 nmol min-' mg protein-', which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein.Purification of the enzyme caused changes in the kinetic properties of the enzyme. The K,,, for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine decreased from 220 pM to 58 pM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V , they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.Tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine : oxygen oxidoreductase (3-hydroxylating)] catalyzes the conversion of tyrosine to 3,4-dihydroxyphenylalanine, which is the initial and rate-limiting step in the biosynthesis of catecholamines such as dopamine, norepinephrine and epinephrine [1,2].

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Section: Discussionmentioning

confidence: 93%

Purification and Some Properties of Tyrosine 3‐Monooxygenase from Rat Adrenal

Okuno

Fujisawa

1982

European Journal of Biochemistry

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“…The EPR signals at g values from 5 to 7 disappear when phenylalanine or pterin are added to the anaerobic enzyme. Since anaerobic incubation of the tetrahydropterin cofactor or phenylalanine with PAH does not result in redox chemistry (Lazarus et al, 1981;Kaufman & Fisher, 1974), the loss of EPR signals cannot furnish information on the prereduction activation (step 1).…”

Section: Discussionmentioning

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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“…These decay rates differ somewhat between the NOS isoforms (Table I) and appear to be faster in full-length NOS compared with the oxygenase domain. Oxidative decay of NOS ferrous heme-O 2 species is increased when H 4 B is bound (35,37,62,63), and the effect is not influenced by substrate. The basis may relate to a redox function for H 4 B (see section below).…”

Section: Minireview: Oxygen Reduction By Nitric-oxide Synthases 14534mentioning

confidence: 92%