Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.
Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.
Mueller
M J
MJ
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Blomster
M
M
Oppermann
U C
UC
Jörnvall
H
H
Samuelsson
B
B
Haeggström
J Z
JZ
D001483:Base Sequence; D018076:DNA, Complementary; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017572:Leukotriene A4 / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics*
D018076:DNA, Complementary; D017572:Leukotriene A4; D011994:Recombinant Proteins; D014443:Tyrosine; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase