Quantifying enzyme activity in living cells

Area of Science:

• Biochemistry
• Cell Biology
• Enzymology

Background:

• Enzymatic activity has traditionally been studied in vitro, assuming cellular conditions mimic these isolated environments.
• The complex and crowded nature of the cellular milieu may significantly alter enzyme kinetics compared to in vitro settings.

Purpose of the Study:

• To determine the real-time catalytic activity of TEM1-β-lactamase within living cells.
• To compare in vivo enzymatic activity with in vitro measurements.
• To investigate the factors influencing enzyme kinetics in the cellular environment.

Main Methods:

• Real-time measurement of TEM1-β-lactamase catalytic activity in living cells.
• Comparison of in vivo kinetic parameters (kcat/Km) with in vitro data.
• Measurement of enzyme and substrate diffusion rates within cells.
• Computational simulations to model reaction dynamics under attenuated diffusion.

Main Results:

• Apparent in vivo catalytic efficiency (kcat/Km) was lower than in vitro values, with considerable cell-to-cell variability.
• In vivo, apparent catalytic efficiency decreased, and Km increased with rising enzyme concentration.
• Substrate diffusion within cells was slower than anticipated, becoming rate-limiting for reactions.
• Simulations indicated substrate flux limitation explains in vivo reaction rates independent of enzyme concentration.

Conclusions:

• In vitro enzymatic data cannot be directly extrapolated to the crowded and complex in vivo cellular environment.
• Substrate diffusion limitations can significantly impact reaction rates in vivo, a factor often overlooked in in vitro studies.
• The findings highlight the need for in vivo studies to accurately understand enzyme function within living systems.