title: A conserved tyrosine residue of Saccharomyces cerevisiae leukotriene A4 hydrolase stabilizes the transition state of the peptidase activity.
abstract: Saccharomyces cerevisiae leukotriene A4 hydrolase (LTA4H) is a bifunctional aminopeptidase/epoxide hydrolase and a member of the M1 family of metallopeptidases. In order to obtain a more thorough understanding of the aminopeptidase activity of the enzyme, two conserved tyrosine residues, Tyr244 and Tyr456, were altered to phenylalanine and the mutant proteins characterized by determining KM and kcat for various amino acid beta-naphthylamide substrates. While mutation of Tyr456 exhibited minimal effect on catalysis, mutation of Tyr244 caused an overall 25-100-fold reduction in catalytic activity for all substrates tested. Furthermore, LTA4H Y244F exhibited a 40-fold decrease in affinity for RB-3014, a transition state analog inhibitor, implicating Tyr244 in transition state stabilization.
authors.affiliation: Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132, USA. mthompso@mtsu.edu
authors.forename: Michael W
authors.initials: MW
authors.lastname: Thompson
authors.forename: Erin D
authors.initials: ED
authors.lastname: Archer
authors.forename: Carrie E
authors.initials: CE
authors.lastname: Romer
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Seipelt
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000378:metabolism*; D007700:Kinetics; D017572:Leukotriene A4 / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D017572:Leukotriene A4; D014443:Tyrosine; D004851:Epoxide Hydrolases; D010447:Peptide Hydrolases; C043134:leukotriene A4 hydrolase