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Probing enzymic transition state hydrophobicities
P P Wangikar1, J O Rich, D S Clark
1Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City 52242, USA.
Biochemistry
|September 26, 1995
Summary
Hydrophobic interactions in enzymes are crucial, even in nonaqueous solvents. Protein engineering and solvent changes can modify these interactions, enabling tailored enzyme function.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Hydrophobic interactions are vital in biological processes but poorly understood in nonaqueous enzymology.
- Understanding these interactions is key to controlling enzyme function in diverse media.
Purpose of the Study:
- To estimate enzyme-substrate hydrophobic interactions for subtilisin BPN' in various solvents.
- To develop a method for quantifying enzymic transition state hydrophobicity.
- To investigate the impact of protein engineering on binding pocket hydrophobicity.
Main Methods:
- Utilized a model reaction catalyzed by subtilisin BPN' in different solvents.
- Employed protein engineering by mutating the Gly166 residue to alter binding pocket hydrophobicity.
- Developed a method to estimate transition state hydrophobicity by varying substrate and solvent properties.
Main Results:
- Enzyme-substrate hydrophobic interactions and transition state stabilization are present in hydrophobic organic solvents.
- Mutating Gly166 to alanine (G166A) and valine (G166V) increased S1 binding pocket hydrophobicity.
- The developed method for calculating binding pocket hydrophobicity is applicable to other enzymes like horseradish peroxidase and alpha-chymotrypsin.
Conclusions:
- Hydrophobic interactions significantly influence enzyme catalysis in both aqueous and nonaqueous environments.
- Protein engineering offers a route to modulate enzyme function by altering binding pocket hydrophobicity.
- The developed method provides a valuable tool for characterizing and tailoring enzyme activity.