| Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase. | |
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MedLine Citation:
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PMID: 23368961 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur. |
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Authors:
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S Colette Daubner; Audrey Avila; Johnathan O Bailey; Dimitrios Barrera; Jaclyn Y Bermudez; David H Giles; Crystal A Khan; Noel Shaheen; Janie Womac Thompson; Jessica Vasquez; Susan P Oxley; Paul F Fitzpatrick |
Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. Date: 2013-02-13 |
Journal Detail:
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Title: Biochemistry Volume: 52 ISSN: 1520-4995 ISO Abbreviation: Biochemistry Publication Date: 2013 Feb |
Date Detail:
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Created Date: 2013-02-27 Completed Date: 2013-04-15 Revised Date: 2013-04-16 |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 1446-55 Citation Subset: IM |
Affiliation:
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Department of Biological Sciences, St. Mary's University, San Antonio, Texas 78228, United States. sdaubner@stmarytx.edu |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Alanine
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genetics,
metabolism Amino Acid Substitution Amino Acids / genetics, metabolism Animals Hydroxylation Models, Molecular Mutagenesis, Site-Directed Phenylalanine Hydroxylase / metabolism* Pterins / metabolism Rats Recombinant Proteins / genetics, metabolism Substrate Specificity Tyrosine 3-Monooxygenase / genetics*, metabolism* |
| Grant Support | |
ID/Acronym/Agency:
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R01 GM047291/GM/NIGMS NIH HHS; R01 GM098140/GM/NIGMS NIH HHS; R25 NS080684/NS/NINDS NIH HHS; R25 NS080684/NS/NINDS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 0/Pterins; 0/Recombinant Proteins; 1008-35-1/tetrahydropterin; 56-41-7/Alanine; EC 1.14.16.1/Phenylalanine Hydroxylase; EC 1.14.16.2/Tyrosine 3-Monooxygenase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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