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 simil...
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Published in | The Journal of biological chemistry Vol. 267; no. 32; pp. 22733 - 22739 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Biochemistry and Molecular Biology
15.11.1992
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Subjects | |
Online Access | Get full text |
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Summary: | "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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/s0021-9258(18)50009-0 |