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Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity
A R Clarke1, C J Smith, K W Hart
1Department of Biochemistry, University of Bristol Medical School, U.K.
Biochemical and Biophysical Research Communications
|October 14, 1987
Summary
Researchers modified the lactate dehydrogenase enzyme from Bacillus stearothermophilus to favor oxaloacetate over pyruvate. This engineered enzyme retains catalytic activity and allosteric regulation, demonstrating successful substrate specificity alteration.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Protein Science
Background:
- Lactate dehydrogenase (LDH) enzymes exhibit substrate specificity for hydroxyacids.
- Understanding enzyme active site determinants is crucial for protein engineering.
Purpose of the Study:
- To alter the substrate specificity of Bacillus stearothermophilus LDH.
- To engineer an enzyme favoring oxaloacetate/malate over pyruvate/lactate.
Main Methods:
- Site-directed mutagenesis was employed to introduce three amino acid substitutions in the LDH gene.
- Specific mutations targeted active site residues to modify substrate binding and catalysis.
Main Results:
- A triple mutant enzyme was created with altered active site volume, neutralized acidity, and introduced basicity.
- The mutant enzyme showed a significant shift in specificity, favoring oxaloacetate over pyruvate by 500-fold, compared to the wild type's 1000-fold preference for pyruvate.
- The engineered malate dehydrogenase retained a catalytic rate constant of 20 s-1 for oxaloacetate reduction and allosteric control by fructose-1,6-bisphosphate.
Conclusions:
- Site-directed mutagenesis can effectively reprogram enzyme substrate specificity.
- Significant alterations to the active site of LDH can yield a functional malate dehydrogenase with retained regulatory properties.