Thiol activation of endopeptidase EC 3.4.24.15. A novel mechanism for the regulation of catalytic activity

Endopeptidase EC 3.4.24.15 (EP24.15) is a thermolysin-like metalloendopeptidase involved in the regulated metabolism of a number of neuropeptides. Unlike other thermolysin-like peptidases EP24.15 displays a unique thiol activation, a mechanism that is not clearly understood. In this study we show th...

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Bibliographic Details
Published in:The Journal of biological chemistry 1997-07, Vol.272 (28), p.17395
Main Authors: Shrimpton, C N, Glucksman, M J, Lew, R A, Tullai, J W, Margulies, E H, Roberts, J L, Smith, A I
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Binding Sites
Catalysis
Cysteine - metabolism
Disulfides - metabolism
Enzyme Activation
Light
Male
Metalloendopeptidases - genetics
Metalloendopeptidases - metabolism
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Conformation
Rats
Rats, Sprague-Dawley
Recombinant Proteins - metabolism
Scattering, Radiation
Sulfhydryl Compounds - metabolism
Testis - enzymology
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Summary:Endopeptidase EC 3.4.24.15 (EP24.15) is a thermolysin-like metalloendopeptidase involved in the regulated metabolism of a number of neuropeptides. Unlike other thermolysin-like peptidases EP24.15 displays a unique thiol activation, a mechanism that is not clearly understood. In this study we show that both recombinant and tissue-derived EP24.15 are activated up to 8-fold by low concentrations (0.1 mM) of dithiothreitol. Additionally, under non-reducing conditions, recombinant and native EP24.15 forms multimers that can be returned to the monomeric form by reduction. We have also shown that competitive inhibitor binding occurs only to the monomeric form, which indicates that catalytic site access is restricted in the multimeric forms. Through systematic site-directed mutagenesis we have identified that cysteine residues 246, 253, and possibly 248 are involved in the formation of these multimers. Furthermore, both a double mutant (C246S/C253S) and a triple mutant (C246S/C248S/C253S) are fully active in the absence of reducing agents, as measured by both inhibitor binding and hydrolysis. The formation and disruption of disulfide bonds involving these cysteine residues may be a mechanism by which EP24.15 activity is regulated through changes in intra- and extracellular redox potential.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.272.28.17395