Mutagenesis of a specificity-determining residue in tyrosine hydroxylase establishes that the enzyme is a robust phenylalanine hydroxylase but a fragile tyrosine hydroxylase.
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.
Department of Biological Sciences, St. Mary's University, San Antonio, Texas 78228, United States. sdaubner@stmarytx.edu
S Colette
SC
Daubner
Audrey
A
Avila
Johnathan O
JO
Bailey
Dimitrios
D
Barrera
Jaclyn Y
JY
Bermudez
David H
DH
Giles
Crystal A
CA
Khan
Noel
N
Shaheen
Janie Womac
JW
Thompson
Jessica
J
Vasquez
Susan P
SP
Oxley
Paul F
PF
Fitzpatrick
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D006900:Hydroxylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D011622:Pterins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism*
D000596:Amino Acids; D011622:Pterins; D011994:Recombinant Proteins; C011552:tetrahydropterin; D010651:Phenylalanine Hydroxylase; D014446:Tyrosine 3-Monooxygenase; D000409:Alanine