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Related Experiment Videos

Long-range structural changes in proteinase K triggered by calcium ion removal.

J Bajorath1, S Raghunathan, W Hinrichs

  • 1Institut für Kristallographie, Freie Universität Berlin, FRG.

Nature
|February 2, 1989
PubMed
Summary

Calcium ions (Ca2+) are crucial for proteinase K activity, even though not directly in catalysis. Removing Ca2+ causes structural changes that reduce enzyme function by altering substrate binding and the catalytic triad.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Proteinase K is a subtilisin-type serine protease.
  • Calcium ions (Ca2+) are known to bind to proteinase K.
  • The precise role of Ca2+ in proteinase K activity and structure is not fully understood.

Purpose of the Study:

  • To elucidate the structural and functional impact of calcium ion (Ca2+) binding and removal on proteinase K.
  • To investigate the mechanism by which Ca2+ influences proteinase K activity.

Main Methods:

  • X-ray crystallography at 1.5 A resolution to determine the structure of proteinase K.
  • Scatchard analysis to quantify Ca2+ binding affinities.
  • Enzyme activity assays using a synthetic substrate (succinyl-Ala-Ala-Ala-p-nitroanilide).

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Main Results:

  • Proteinase K possesses two Ca2+ binding sites: one high-affinity and one low-affinity.
  • Ca2+ is not directly part of the catalytic triad (Asp39-His69-Ser224).
  • Removal of Ca2+ from the high-affinity site induces significant structural rearrangements in peripheral loops and alpha-helices, affecting the catalytic triad and substrate binding site, leading to a ~80% loss of activity.

Conclusions:

  • Calcium ions (Ca2+) play a critical allosteric role in maintaining the structural integrity and optimal function of proteinase K.
  • The observed structural changes upon Ca2+ depletion explain the reduction in enzyme activity, highlighting an indirect mechanism of regulation.