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Related Experiment Videos

Determining appropriate substrate conversion for enzymatic assays in high-throughput screening.

Ge Wu1, Yue Yuan, C Nicholas Hodge

  • 1Amphora Discovery Corp. Los Altos, CA, USA. ge.wu@fiveprime.com

Journal of Biomolecular Screening
|January 9, 2004
PubMed
Summary

High substrate conversion in enzyme kinetics assays improves signal but may reduce inhibitor sensitivity. This study models the relationship between substrate conversion and IC(50) values, guiding assay design for better high-throughput screening (HTS).

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Assay development

Background:

  • Enzyme kinetics studies traditionally recommend <10% substrate conversion for accuracy.
  • High-throughput screening (HTS) often requires higher substrate conversion (>10%) for sufficient signal-to-background ratios.
  • The impact of high substrate conversion on assay sensitivity and screening reliability is not well understood.

Purpose of the Study:

  • To investigate the consequences of high substrate conversion on enzymatic assay sensitivity.
  • To derive a theoretical relationship between IC(50) values and substrate conversion percentage.
  • To provide guidance for optimizing substrate conversion in HTS assay design.

Main Methods:

  • Developed a theoretical model based on first-order kinetics under Henri-Michaelis-Menten conditions.

Related Experiment Videos

  • Derived the relationship between IC(50) and substrate conversion percentage.
  • Experimentally validated the model using cAMP-dependent protein kinase.

    • Main Results:

    • A mathematical model was established to predict the effect of substrate conversion on IC(50) values.
    • Experimental results with cAMP-dependent protein kinase confirmed the theoretical predictions.
    • The study quantifies the trade-off between signal robustness and inhibitor sensitivity at varying substrate conversion levels.

    Conclusions:

    • High substrate conversion can compromise assay sensitivity, impacting the accuracy of IC(50) determination.
    • The derived model allows assay developers to select optimal substrate conversion for HTS.
    • Balancing signal strength and inhibitor sensitivity is crucial for robust and reliable enzymatic assays.