Related Experiment Videos
Crystal structures of two engineered thiol trypsins.
M E McGrath1, M E Wilke, J N Higaki
1Department of Biochemistry, University of California, San Francisco 94143-0448.
Biochemistry
|November 28, 1989
Summary
Engineered trypsins with cysteine replacing serine reveal structural insights into cysteine protease activity. These studies explain significant activity loss in modified enzymes, offering new perspectives on protease mechanisms.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Serine proteases and cysteine proteases are distinct enzyme classes with different catalytic mechanisms.
- Engineering serine proteases to mimic cysteine proteases provides a unique system to study enzyme function.
Purpose of the Study:
- To determine the three-dimensional structures of engineered rat trypsins mimicking cysteine protease active sites.
- To elucidate the structural basis for reduced enzymatic activity in these engineered proteases.
Main Methods:
- X-ray crystallography was used to solve the structures of engineered rat trypsins (S195C and D102N,S195C) at high resolution.
- Structural analysis focused on active site residues, hydrogen bonding networks, and overall enzyme conformation.
Main Results:
- The structures revealed reduced active site thiols, unlike previously observed oxidized thiols in thiol proteases.
- Mutations led to significant losses in enzymatic activity (10^6-fold for S195C, 10^8-fold for D102N,S195C).
- The D102N mutation altered the hydrogen bonding of histidine 57, hindering its ability to act as a catalytic base.
Conclusions:
- These are the first structures of serine proteases engineered to possess cysteine protease catalytic centers.
- Subtle changes in active site hydrogen bonding, particularly involving residue 102, are responsible for the dramatic loss of activity.
- The findings provide critical insights into the catalytic mechanisms of both serine and cysteine proteases.