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Enzyme crystal structure in a neat organic solvent
P A Fitzpatrick1, A C Steinmetz, D Ringe
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Summary
The crystal structure of subtilisin Carlsberg in anhydrous acetonitrile is nearly identical to its structure in water. Most structural water molecules remain bound, while acetonitrile occupies active site regions.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Subtilisin Carlsberg is a well-studied serine protease.
- Enzyme structure and function are sensitive to solvent environment.
- Understanding enzyme behavior in non-aqueous solvents is crucial for biocatalysis.
Purpose of the Study:
- To determine the crystal structure of subtilisin Carlsberg in anhydrous acetonitrile.
- To compare the enzyme's structure in acetonitrile to its aqueous structure.
- To investigate the binding of acetonitrile and the role of structural water molecules.
Main Methods:
- X-ray crystallography at 2.3 A resolution.
- Structural comparison with previously determined enzyme structures.
- Analysis of solvent molecule interactions.
Main Results:
- The overall three-dimensional structure in acetonitrile is highly similar to that in water.
- The catalytic triad's hydrogen bond system remains intact.
- Most (99/119) structural water molecules are retained.
- Acetonitrile molecules bind, with some displacing water and others binding to new sites.
- One-third of bound acetonitrile molecules occupy active site regions (P1, P2, P3).
Conclusions:
- Subtilisin Carlsberg maintains its core structure and catalytic machinery in anhydrous acetonitrile.
- Enzyme-bound water molecules exhibit high affinity and are largely retained.
- Acetonitrile can bind within the active site, potentially influencing substrate binding.