Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme

Akira Nakashima, Keiji Mori, Takahiro Suzuki, Hideki Kurita, Motohiko Otani, Toshiaru Nagatsu, Akira Ota

研究成果: Article

35 引用 (Scopus)

抄録

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus- deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(L-erythro-1',2'- dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N- terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del- 38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence Gly36-Arg37-Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.

元の言語English
ページ(範囲)2145-2153
ページ数9
ジャーナルJournal of Neurochemistry
72
発行部数5
DOI
出版物ステータスPublished - 28-04-1999

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Tyrosine 3-Monooxygenase
Dopamine
Enzymes
Amino Acids
Catecholamines
Tyrosine
Iron
Maltose-Binding Proteins
Size exclusion chromatography
Biosynthesis
Escherichia coli
Gel Chromatography
Amino Acid Sequence
Catalyst activity
Fusion reactions
Feedback

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cellular and Molecular Neuroscience

これを引用

Nakashima, Akira ; Mori, Keiji ; Suzuki, Takahiro ; Kurita, Hideki ; Otani, Motohiko ; Nagatsu, Toshiaru ; Ota, Akira. / Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme. :: Journal of Neurochemistry. 1999 ; 巻 72, 番号 5. pp. 2145-2153.
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abstract = "Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus- deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(L-erythro-1',2'- dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N- terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del- 38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence Gly36-Arg37-Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.",
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Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme. / Nakashima, Akira; Mori, Keiji; Suzuki, Takahiro; Kurita, Hideki; Otani, Motohiko; Nagatsu, Toshiaru; Ota, Akira.

:: Journal of Neurochemistry, 巻 72, 番号 5, 28.04.1999, p. 2145-2153.

研究成果: Article

TY - JOUR

T1 - Dopamine inhibition of human tyrosine hydroxylase type 1 is controlled by the specific portion in the N-terminus of the enzyme

AU - Nakashima, Akira

AU - Mori, Keiji

AU - Suzuki, Takahiro

AU - Kurita, Hideki

AU - Otani, Motohiko

AU - Nagatsu, Toshiaru

AU - Ota, Akira

PY - 1999/4/28

Y1 - 1999/4/28

N2 - Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus- deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(L-erythro-1',2'- dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N- terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del- 38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence Gly36-Arg37-Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.

AB - Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus- deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(L-erythro-1',2'- dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N- terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del- 38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence Gly36-Arg37-Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.

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