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Intramolecular processing of prothermolysin

C Marie-Claire1, B P Roques, A Beaumont

  • 1Departement de Pharmacochimie Moleculaire and Structurale, U266 INSERM URA D1500 CNRS, UER des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l'Observatoire, 75270 Paris Cedex 06, France.

The Journal of Biological Chemistry
|April 16, 1998
PubMed
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The prosequence of thermolysin (an extracellular zinc endopeptidase) aids refolding and inhibits activity. Its cleavage from the mature enzyme occurs via an autocatalytic intramolecular pathway, as demonstrated by mutagenesis studies.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Thermolysin, a zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme.
  • The prosequence is known to assist in vitro refolding and inhibit enzyme activity.

Purpose of the Study:

  • To investigate if prosequence cleavage from mature thermolysin is autocatalytic.
  • To determine if this cleavage process is intermolecular or intramolecular.

Main Methods:

  • Expression of N-terminal histidine-tagged prothermolysin in Escherichia coli.
  • Solubilization, immobilization on a resin, and renaturation of prothermolysin.
  • Introduction of mutations to inactivate mature thermolysin activity.

Main Results:

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  • Partial processing to mature enzyme was observed in vivo, with most proenzyme recovered intact.
  • Solubilized and renatured prothermolysin was quantitatively transformed to mature enzyme.
  • Mutated prothermolysin failed to be processed, both in vivo and in vitro, even with exogenous thermolysin.

Conclusions:

  • Thermolysin maturation involves autocatalytic cleavage of the prosequence.
  • The cleavage mechanism is demonstrated to be an intramolecular process.