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A conserved tryptophan residue with an altered pKa is essential for the peptidase reaction of Saccharomyces cerevisiae leukotriene A4 hydrolase
Author(s) -
Archer Erin,
Seipelt Rebecca L.,
Thompson Michael W.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a471-c
Subject(s) - biochemistry , enzyme , aminopeptidase , enzyme kinetics , tryptophan , chemistry , alanine , leucine , hydrolase , residue (chemistry) , saccharomyces cerevisiae , enzyme assay , amino acid , active site , stereochemistry , yeast
Leukotriene A 4 hydrolase is an unusual bifunctional enzyme with both an epoxide hydrolase activity and an aminopeptidase activity. In order to obtain a better understanding of the molecular mechanism of the peptidase activity of the enzyme, a highly conserved tryptophan residue (W356) was mutated to leucine and a conserved aspartate residue (D399) was mutated to alanine. Enzymatic function of W356L and D399A were investigated using amino acid β‐naphthylamide substrates to characterize its substrate specificity and specific activity. While mutation of D399 had no discernable effect, mutation of W356 lowered the k cat for the enzyme 30 to 400‐fold for most substrates tested, with little to no effect on the affinity of the enzyme for the substrates tested. Additionally, the W356L mutation also greatly altered the pH sensitivity of the enzyme without affecting enzyme structure, indicating that W356 most likely exhibits an altered pKa that may be essential to its role in the catalytic mechanism.