Document Detail


Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase.
MedLine Citation:
PMID:  23368961     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
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:
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:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-27     Completed Date:  2013-04-15     Revised Date:  2014-04-08    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1446-55     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Alanine / 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:
R01 GM047291/GM/NIGMS NIH HHS; R01 GM047291/GM/NIGMS NIH HHS; R25 NS080684/NS/NINDS NIH HHS; R25 NS080684/NS/NINDS NIH HHS
Chemical
Reg. No./Substance:
0/Amino Acids; 0/Pterins; 0/Recombinant Proteins; 1008-35-1/tetrahydropterin; EC 1.14.16.1/Phenylalanine Hydroxylase; EC 1.14.16.2/Tyrosine 3-Monooxygenase; OF5P57N2ZX/Alanine
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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