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Related Experiment Video

Updated: Feb 25, 2026

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues
08:36

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues

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Quantifying enzyme activity in living cells.

Agnes Zotter1, Felix Bäuerle1,2, Debabrata Dey1

  • 1From the Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel and.

The Journal of Biological Chemistry
|August 9, 2017
PubMed
Summary

Enzyme activity measured inside living cells differs significantly from in vitro studies. Cellular environments can limit reaction rates, making in vitro data unreliable for predicting in vivo enzyme function.

Keywords:
Michaelis–Mentenbeta-lactamasebiophysicsenzymeenzyme kineticsin vivo imaging

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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.