Mechanism-based inactivation of leukotriene A4 hydrolase/aminopeptidase by leukotriene A4. Mass spectrometric and kinetic characterization.
"Suicide" inactivation of leukotriene (LT) A4 hydrolase/aminopeptidase occurs via an irreversible mechanism-based process which is saturable, of pseudo firstorder, and dependent upon catalysis. Data obtained with either recombinant enzyme or enzyme purified from human leukocytes were similar. Apparent binding constants and inactivation rate constants are equivalent, compatible with a single type of substrate-enzyme complex which partitions between two fates, turnover and inactivation. Both catalytic functions are inactivated, consistent with an overlapping active site for this bifunctional enzyme. The partition ratio (turnover/inactivation) for the LTA4-enzyme complex is 129 +/- 16 for LTA4 hydrolase activity and 124 +/- 10 for aminopeptidase activity. The pH dependence for turnover and inactivation are indistinguishable with a maximum at pH 8. L-Proline p-nitroanilide, a weak substrate with a high Km for the aminopeptidase affords only partial protection against inactivation by LTA4. However, two potent competitive inhibitors, bestatin and captopril, protect both catalytic processes from inactivation, consistent with an active-site specificity for the suicide event. Electrospray ionization mass spectrometry indicates that the molecular weight of pure recombinant enzyme is 69,399 +/- 4 and that covalent modification accompanies catalysis, producing an LTA4:enzyme adduct with a molecular weight 69,717 +/- 4 and a 1:1 stoichiometry. In agreement with kinetic data, electrospray ionization mass spectrometry shows that bestatin inhibits the covalent modification of enzyme by LTA4 and that the extent of modification is proportional to the loss of enzymatic activity.
Orning
L
L
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Gierse
J
J
Duffin
K
K
Bild
G
G
Krivi
G
G
Fitzpatrick
F A
FA
D001667:Binding, Competitive; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors* / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology; D017572:Leukotriene A4; D015289:Leukotrienes / Q000494:pharmacology*; D013058:Mass Spectrometry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013379:Substrate Specificity
D017572:Leukotriene A4; D015289:Leukotrienes; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase