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Monomeric human cathepsin E
1School of Molecular and Medical Biosciences, University of Wales College of Cardiff, UK.
FEBS Letters
|June 5, 1995
Summary
Human Cathepsin E, a proteinase, normally forms a dimer. Mutating a key cysteine residue created a monomeric form, which retained activity but lost stability at high pH and temperature.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Cathepsin E is a aspartic proteinase known to exist as a homodimer.
- The homodimerization is mediated by an inter-molecular disulphide bond involving a specific cysteine residue near the N-terminus.
Purpose of the Study:
- To investigate the role of the inter-molecular disulphide bond in Cathepsin E structure and function.
- To engineer and characterize a monomeric mutant of human Cathepsin E.
Main Methods:
- Site-directed mutagenesis was used to replace the cysteine residue with alanine.
- The engineered mutant protein was purified.
- Enzyme activity was assessed using chromogenic peptide substrates.
- Enzyme susceptibility to pepstatin inhibition was evaluated.
- Protein stability was tested under alkaline pH and elevated temperature conditions.
Main Results:
- A monomeric form of human Cathepsin E was successfully engineered and purified.
- The monomeric mutant exhibited comparable hydrolytic activity against peptide substrates.
- Pepstatin inhibition profiles remained similar between the wild-type and mutant enzymes.
- The monomeric Cathepsin E displayed significantly reduced stability towards alkaline pH and heat.
Conclusions:
- The inter-molecular disulphide bond is crucial for the stability of Cathepsin E.
- While essential for stability, the disulphide bond does not appear to be critical for the catalytic activity or pepstatin inhibition of Cathepsin E.