Mutagenesis of a Specificity-Determining Residue in Tyrosine Hydroxylase Establishes That the Enzyme Is a Robust Phenylalanine Hydroxylase but a Fragile Tyrosine Hydroxylase

详细信息    查看全文

• 作者：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
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：February 26, 2013
• 年：2013
• 卷：52
• 期：8
• 页码：1446-1455
• 全文大小：390K
• 年卷期：v.52,no.8(February 26, 2013)
• ISSN：1520-4995

文摘

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鈥?661]. Alanine-scanning mutagenesis of amino acids 423鈥?27, 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 10p>4p>) 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.