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Identification of Gln 313 and Pro 327 as Residues Critical for Substrate Inhibition in Tyrosine Hydroxylase
Author(s) -
Quinsey Noelene S.,
Lenaghan Catherine M.,
Dickson Phillip W.
Publication year - 1996
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1996.66030908.x
Subject(s) - pterin , phenylalanine hydroxylase , tyrosine hydroxylase , phenylalanine , tyrosine , tetrahydrobiopterin , chemistry , tyrosine 3 monooxygenase , leucine , biochemistry , alanine , stereochemistry , enzyme , amino acid , cofactor
Rat tyrosine hydroxylase was expressed in Escherichia coli . High‐level expression was obtained after incubation at 27°C for 18 h. The smallest fragment of tyrosine hydroxylase that gave a soluble active molecule was from Leu 188 to Phe 456 . 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 Glu 286 plays a critical role in pterin function in phenylalanine hydroxylase. The corresponding residue in tyrosine hydroxylase (Glu 332 ) 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 (Pro 281 ) 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 Pro 327 or a glutamic acid for Gln 313 in tyrosine hydroxylase eliminates the substrate inhibition shown by wild‐type tyrosine hydroxylase.