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Inhibition of cathepsin E by alpha 2-macroglobulin and the resulting structural changes in the inhibitor
S B Athauda1, H Arakawa, M Nishigai
1Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo.
Abstract:
The activity of cathepsin E (M(r) approximately 80K), a dimeric aspartic proteinase with two active sites per molecule, toward a protein substrate (reduced and carboxymethylated ribonuclease A) was shown to be completely inhibited by alpha 2-macroglobulin (alpha 2M) at pH 5.5. On the other hand, the activity toward a peptide substrate (oxidized insulin B chain) was scarcely inhibited. Under these conditions, cathepsin E cleaved alpha 2M at the Phe684-Tyr685 bond in the bait region sequence, resulting in a drastic conformational change (from a doughnut to an H shape) in the inhibitor as revealed by electron microscopy, and was non-covalently trapped by alpha 2M in an approximate molar ratio (enzyme: alpha 2M) of 2:1.
Insights
Cathepsin E activity against protein substrates is fully inhibited by alpha 2-macroglobulin (alpha 2M). However, peptide substrate activity is minimally affected, with cathepsin E cleaving alpha 2M.
Area of Science:
- Biochemistry
- Enzymology
- Proteomics
Background:
- Cathepsin E is a dimeric aspartic proteinase with two active sites.
- Alpha 2-macroglobulin (alpha 2M) is a major plasma proteinase inhibitor.
- Understanding enzyme-inhibitor interactions is crucial for molecular biology.
Purpose of the Study:
- To investigate the inhibitory activity of alpha 2-macroglobulin (alpha 2M) against cathepsin E.
- To characterize the interaction between cathepsin E and alpha 2M at a molecular level.
- To determine the substrate specificity of cathepsin E inhibition by alpha 2M.
Main Methods:
- Enzyme activity assays using protein and peptide substrates.
- Inhibition studies with alpha 2-macroglobulin (alpha 2M).
- Analysis of alpha 2M cleavage and conformational changes using electron microscopy.
Main Results:
- Cathepsin E's activity on protein substrates was completely inhibited by alpha 2M at pH 5.5.
- Cathepsin E's activity on peptide substrates showed minimal inhibition.
- Alpha 2M underwent a conformational change and trapped cathepsin E in a 2:1 molar ratio after cleavage at the Phe684-Tyr685 bond.
Conclusions:
- Cathepsin E exhibits differential inhibition by alpha 2M based on substrate type.
- Alpha 2M effectively inhibits cathepsin E's proteinase activity through cleavage and conformational trapping.
- The findings provide insights into the specific molecular interactions between cathepsin E and alpha 2M.