A general method for relieving substrate inhibition in lactate dehydrogenases

Area of Science:

• Biochemistry
• Enzyme Engineering
• Molecular Biology

Background:

• Substrate inhibition in lactate dehydrogenase (LDH) enzymes can limit their industrial utility.
• Understanding the molecular basis of substrate inhibition is crucial for enzyme engineering.

Purpose of the Study:

• To investigate the effects of the S163L mutation on human heart lactate dehydrogenase (LDH) activity.
• To determine if the S163L mutation can be a general strategy for removing substrate inhibition in LDH enzymes.
• To explore the industrial applications of engineered LDH with removed substrate inhibition.

Main Methods:

• Site-directed mutagenesis to introduce the S163L mutation.
• Enzyme kinetics assays to measure turnover rates and Michaelis constants (KM).
• Nuclear Magnetic Resonance (NMR) spectroscopy to study cofactor binding.
• Molecular modeling to visualize enzyme-cofactor complexes.

Main Results:

• The S163L mutation effectively removed substrate inhibition by preventing the formation of a covalent adduct between pyruvate and the cofactor.
• The mutation caused a modest reduction in turnover rate but a significant increase in the KM for pyruvate.
• NMR and molecular modeling indicated a non-productive bound form of the cofactor in the S163L mutant.

Conclusions:

• The S163L mutation is a viable strategy for eliminating substrate inhibition in human heart LDH.
• Engineered LDH enzymes with removed substrate inhibition have potential industrial applications, particularly in the synthesis of alpha-hydroxy acids.
• A mechanism involving a non-productive cofactor complex explains the observed kinetic properties of the S163L mutant.