Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells

Fukami, Miriam H.; Haavik, Jan; Flatmark, Torgeir

doi:10.1042/bj2680525

Cited by 39 publications

(23 citation statements)

References 22 publications

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“…When phenylalanine is used as a substrate for TYH, the major product is tyrosine , but rn-hydroxyphenylalanine is also formed at T low levels (Fukami et al, 1990). This is the result expected if an arene oxide is formed initially (Fig.…”

Section: Recent Mechanistic Studies Of Tyrosine and Tryptophan Hydmentioning

confidence: 63%

The Aromatic Amino Acid Hydroxylases

Fitzpatrick

2000

Advances in Enzymology - And Related Areas of Molecular Biology

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Section: Recent Mechanistic Studies Of Tyrosine and Tryptophan Hydmentioning

confidence: 63%

The Aromatic Amino Acid Hydroxylases

Fitzpatrick

2000

Advances in Enzymology - And Related Areas of Molecular Biology

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“…1976) and synaptosomal preparations (Katz et al . 1976), PC12 cells (De Pietro and Fernstrom 1999), and bovine chromaffin cells (Fukami et al . 1990) resulted in the synthesis of DOPA from the radioactive Phe.…”

Section: Discussionmentioning

confidence: 99%

Relationship between myelin production and dopamine synthesis in the PKU mouse brain

Joseph

Dyer

2003

Journal of Neurochemistry

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Phenylketonuria is caused by specific mutations in the phenylalanine hydroxylase gene and is characterized by elevated blood phenylalanine levels, hypomyelination in forebrain structures, reduced dopamine levels, and cognitive difficulties. To determine whether brain tyrosine levels and/or myelination play a role in the up-regulation of dopamine, phenylketonuric mice were placed on a low phenylalanine diet for 4 weeks and as blood phenylalanine levels dropped to normal, the relationships between phenylalanine, tyrosine, dopamine, myelin proteins, and axonal proteins in frontal cortex and striatum were determined using gas chromatography mass spectrometry, histology, and western blotting techniques. Blood phenylalanine rapidly decreased from an eight-fold elevation to near control levels, and blood tyrosine gradually rose from about 50% to near normal values. In frontal cortex and striatum, phenylalanine levels dropped to 2-and 1.5-fold elevations above control, respectively, and tyrosine levels increased but remained less than 70% of control in both structures. In frontal cortex, increases in dopamine and myelin basic protein occurred in a similar biphasic pattern, reaching near normal levels by week 4. In striatum, dopamine and MBP dramatically increased to near normal levels in the first week. Myelination was confirmed histologically and by western blot quantification of phosphorylated neurofilaments. In summary, our results showed: (i) an increase in dopamine despite low brain tyrosine levels and (ii) similar recovery patterns for myelination and dopamine. Since myelin/axonal interactions trigger signaling pathways that result in axonal maturation, we speculate that this interaction also may trigger signals that up-regulate neurotransmitter synthesis. Keywords: axon, blood-brain barrier, dopamine, myelin, phenylketonuria, tyrosine. In the CNS, oligodendrocytes extend numerous processes, and from the distal tip of each process, a membrane sheet is assembled and wrapped around a segment of axon as an internode of myelin. Myelin is a highly metabolically active membrane that, under normal conditions, remains connected to and is supported by the oligodendrocyte cell body for the life span of the oligodendrocyte. Myelin is essential for the rapid conduction of action potentials and, therefore, there may be devastating consequences if oligodendrocytes fail to produce myelin (hypomyelination) or lose their myelin (demyelination) as a consequence of disease.One disease in which hypomyelination occurs in specific forebrain tracts, but neurons and their axons are spared, is the autosomal recessive disorder phenylketonuria (PKU) (Malamud 1966;Dyer et al. 1996). PKU is caused by a rise in blood phenylalanine (Phe) levels, due to a deficiency in the enzyme phenylalanine hydroxylase (PAH) (Jervis 1953). PAH is expressed primarily in liver and not in brain, and catalyzes the conversion of Phe to tyrosine (Hsieh and Berry 1979). Blood Phe levels normally are about 121 lM; however, in untreated individuals (and mice) ...

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“…Other than release from cooperative substrate activation of PAH, removal of the regulatory domains of PAH and TYH does not affect the ability of these enzymes to hydroxylate the other aromatic amino acids (17). TYH will hydroxylate phenylalanine (48,49). Whereas the rate of tetrahydropterin oxidation by TYH in the presence of phenylalanine is comparable to that seen in the presence of tyrosine, only a fraction of the reducing equivalents are used to form tyrosine (17,49).…”

Section: Discussionmentioning

confidence: 99%

Expression and Characterization of the Catalytic Core of Tryptophan Hydroxylase

Moran¹,

Daubner²,

Fitzpatrick³

1998

Journal of Biological Chemistry

107

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Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH 102-416 , was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH 102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4a-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.Tryptophan hydroxylase (TRH 1 , EC 1.14.16.4) carries out the 5-hydroxylation of tryptophan via the oxidation of tetrahydropterin and the reductive incorporation of molecular oxygen (Scheme 1). In mammalian metabolism the reaction catalyzed by TRH precedes ␣-decarboxylation and is believed to be the initial and rate-limiting process in the production of the neurotransmitter serotonin (5-hydroxytryptamine). Although TRH has been studied since the early 70s, enzymological characterization has been impeded by the limited quantity of active enzyme available from native or heterologous sources, the exceedingly low specific activity of the isolated enzyme, and the quite rapid decrease in activity observed during purification or storage (1-9).TRH is a member of the small family of pterin-dependent aromatic amino acid hydroxylases that includes tyrosine hydroxylase (TYH) and phenylalanine hydroxylase (PAH). Each of these enzymes catalyzes the addition of an oxygen atom to the ring of an aromatic amino acid substrate. The bulk of what is currently known of the reaction mechanism of these enzymes has come from studies of the latter two (10). Both PAH and TYH require ferrous iron for activity (11,12); however, the exact role for the iron in catalysis is undefined. The primary structures of these enzymes are known from a variety of organisms. Sequence comparisons and deletion mutageneses have identified three functional regions: an amino-terminal regulatory domain, a catalytic domain, and a carboxyl-terminal interface (13-17). The regulatory domains of the three hydroxylases show no similarities, whereas the catalytic domains are homologous, with sequence identities of 32-75%. Enzymes lacking the regulatory domain are catalytically active (13,14,17). The carboxyl-terminal 24 amino acids of TYH form a long helix demonstrated to be responsible for the tetrameric structure of the enzyme (13, 18); this helix is presumed to have a corresponding function in TRH and PAH.We report here the purification and preliminary characterization of a mutant protein containing only the catalytic core of TRH. The rationale for the truncations w...

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Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells

Cited by 39 publications

References 22 publications

The Aromatic Amino Acid Hydroxylases

The Aromatic Amino Acid Hydroxylases

Relationship between myelin production and dopamine synthesis in the PKU mouse brain

Expression and Characterization of the Catalytic Core of Tryptophan Hydroxylase

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