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Sulfur shuffle: modulating enzymatic activity by thiol-disulfide interchange.

J M Messmore1, S K Holmgren, J E Grilley

  • 1Department of Biochemistry and Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.

Bioconjugate Chemistry
|May 23, 2000
PubMed
Summary

Researchers developed a general strategy to control enzyme activity using modified cysteine residues. This method, demonstrated with ribonuclease A (RNase A), allows for reversible modulation of catalytic function by forming specific disulfide bonds.

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Chemical biology

Background:

  • Enzyme activity modulation is crucial for biological chemistry.
  • Ribonuclease A (RNase A) cleaves RNA, with Lys41 playing a key role in catalysis.
  • Replacing Lys41 with cysteine significantly reduces RNase A activity.

Purpose of the Study:

  • To present a general strategy for controlling enzyme catalytic activity.
  • To demonstrate this strategy using a modified ribonuclease A enzyme.
  • To explore reversible control of enzyme function via disulfide bond formation.

Main Methods:

  • Replacing Lys41 in RNase A with cysteine (K41C RNase A).
  • Forming mixed disulfides between Cys41 and cysteamine to restore activity.
  • Further modifying the disulfide with mercaptopropyl phosphate to inhibit activity.

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  • Using dithiothreitol for disulfide bond reduction and inorganic phosphate for reactivation.

    • Main Results:

    • K41C RNase A showed a 10^5-fold decrease in catalytic efficiency (kcat/Km).
    • Mixed disulfide formation with cysteamine restored activity by 10^3-fold.
    • Mercaptopropyl phosphate modification decreased activity an additional 25-fold.
    • Enzyme activity was reversibly controlled by disulfide bond manipulation.

    Conclusions:

    • A general strategy for enzyme activity control via cysteine modification and disulfide bonds is established.
    • This method allows for facile and reversible modulation of enzymatic catalysis.
    • The strategy is potentially applicable to a wide range of enzymes by targeting essential residues.