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Related Experiment Videos

Papain-catalyzed reactions at subzero temperatures

A L Fink, K J Angelides

    Biochemistry
    |November 30, 1976
    PubMed
    Summary
    This summary is machine-generated.

    Investigating papain catalysis at subzero temperatures, this study found that dimethyl sulfoxide and ethanol are suitable cryosolvents. Enzyme-substrate intermediates were successfully trapped and characterized at low temperatures.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Cryochemistry

    Background:

    • Papain catalysis is crucial for various biochemical processes.
    • Understanding enzyme-substrate intermediates aids in elucidating reaction mechanisms.
    • Subzero temperatures offer a unique environment for studying transient intermediates.

    Purpose of the Study:

    • To investigate papain catalysis at subzero temperatures.
    • To detect, accumulate, and characterize enzyme-substrate intermediates.
    • To evaluate the suitability of cryosolvents like dimethyl sulfoxide and ethanol.

    Main Methods:

    • Studied papain-catalyzed hydrolysis of Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester.
    • Utilized subzero temperatures and aqueous dimethyl sulfoxide solutions.

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  • Performed preliminary intermediate trapping experiments with Nalpha-carbobenzoxy-L-lysine methyl ester.

    • Main Results:

    • Dimethyl sulfoxide (DMSO) concentration affects kinetic parameters (kcat, Km) but does not cause structural changes in papain.
    • Arrhenius plots showed linearity for deacylation in 7.65 M DMSO from 0 to -45°C.
    • Acyl enzyme intermediates were successfully trapped below -50°C, with 1:1 stoichiometry.
    • Aqueous ethanol solutions up to 80% (v/v) were also suitable cryosolvents.
    • Preliminary experiments suggest three detectable enzyme-substrate intermediates.

    Conclusions:

    • 7.65 M DMSO and subzero temperatures are suitable for papain catalysis without deleterious effects.
    • Subzero temperatures enable the accumulation and characterization of papain-enzyme intermediates.
    • Aqueous ethanol is also a viable cryosolvent for papain studies.