Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Enzyme activity is often modulated by subunit composition.
• Understanding protein expression and function at the single-molecule level is crucial.
• Mistranslation, errors in protein synthesis, can impact enzyme kinetics.

Purpose of the Study:

• To engineer and characterize tetrameric beta-galactosidase (β-gal) with variable numbers of active monomers.
• To investigate enzyme kinetics at the single-molecule level for different β-gal populations.
• To accurately measure in vitro mistranslation frequency using single-molecule enzyme kinetics.

Main Methods:

• Engineered expression of tetrameric β-gal by combining wild-type and single-nucleotide polymorphism plasmids.
• Utilized single-molecule-level enzyme kinetics to analyze enzyme populations.
• Measured in vitro mistranslation frequency.

Main Results:

• Successfully expressed tetrameric β-gal with one to four active monomers in vitro.
• Identified four distinct enzyme kinetic populations corresponding to the number of active monomers.
• Determined an accurate in vitro mistranslation frequency of 5.8 × 10^-4 per base.
• Characterized the kinetics of mistranslated β-gal at the single-molecule level.

Conclusions:

• The number of active monomers in tetrameric β-gal directly influences its enzyme kinetics.
• Single-molecule enzyme kinetics is a powerful tool for dissecting enzyme heterogeneity.
• This study provides a precise measurement of in vitro mistranslation frequency and its impact on enzyme function.