Measurement of kon without a rapid-mixing device

Authors

  • James Kahn,

    1. Department of Chemistry and Biochemistry, University of San Diego, San Diego, California 92110
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  • Robert N. Dutnall,

    1. Department of Chemistry and Biochemistry, University of San Diego, San Diego, California 92110
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  • Kimberly Matulef,

    1. Department of Chemistry and Biochemistry, University of San Diego, San Diego, California 92110
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  • Leigh A. Plesniak

    Corresponding author
    1. Department of Chemistry and Biochemistry, University of San Diego, San Diego, California 92110
    2. Department of Biology, University of San Diego, San Diego, California 92110
    • Department of Chemistry and Biochemistry, University of San Diego, San Diego, California 92110
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    • Tel.: 859-692-0460; Fax: 619-260-6804


  • This work is supported by NIH AREA grant GM068431-02A1.

Abstract

We have recently designed a biochemistry laboratory experiment for the purpose of providing students an advanced experience with enzyme kinetics and the kinetics of binding. Bestatin, a well-known and commercially available general protease inhibitor, is a slow-binding inhibitor of aminopeptidase isolated from Aeromonas proteolytica. The binding is on a timescale slow enough for measurement without the use of a rapid-mixing device. Aminopeptidase inhibition is detected via a standard colorimetric assay with an inexpensive commercially available substrate. The binding of bestatin follows first order binding kinetics with a rate constant kon of 59 ± 5 M−1 s−1. This aminopeptidase is well characterized with several crystal structures and a published Ki, which students can then use to calculate the value for koff.

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