Identification of Gln313 and Pro327 as residues critical for substrate inhibition in tyrosine hydroxylase.
Rat tyrosine hydroxylase was expressed in Escherichia coli. High-level expression was obtained after incubation at 27 degrees C for 18 h. The smallest fragment of tyrosine hydroxylase that gave a soluble active molecule was from Leu188 to Phe456. This fragment corresponds directly to the section of phenylalanine hydroxylase that had previously been shown to be this enzyme's catalytic core region. It has been shown that Glu288 plays a critical role in pterin function in phenylalanine hydroxylase. The corresponding residue in tyrosine hydroxylase (Glu332) has no significant role in pterin function. Substitution of a leucine for a proline at position 327 in tyrosine hydroxylase produces a molecule with a K(m) for tetrahydrobiopterin 20-fold higher than that of the wild-type molecule, whereas the same substitution at the corresponding residue in phenylalanine hydroxylase (pro281) has no effect on the kinetic constant for the cofactor. This suggests that corresponding residues in phenylalanine hydroxylase and tyrosine hydroxylase can have different roles in pterin function. Substitution of a leucine for a proline at position 281 in phenylalanine hydroxylase increases the K(m) for phenylalanine > 20-fold over that of the wild-type. Substitution of leucine or alanine for Pro327 or a glutamic acid for Gln313 in tyrosine hydroxylase eliminates the substrate inhibition shown by wild-type tyrosine hydroxylase.
Quinsey
N S
NS
Russell Grimwade Laboratory of Protein Engineering, Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.
Lenaghan
C M
CM
Dickson
P W
PW
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000235:genetics; D051381:Rats; D011995:Recombination, Genetic; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism*
D010446:Peptide Fragments; D010651:Phenylalanine Hydroxylase; D014446:Tyrosine 3-Monooxygenase