Intramolecular processing of prothermolysin.
Thermolysin, an extracellular zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O'Donohue, M. J., and Beaumont, A. (1996) J. Biol. Chem. 271, 26477-26481). To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, N-terminal histidine-tagged prothermolysin was expressed in Escherichia coli. Although partial processing to mature enzyme occurred, most of the proenzyme was recovered intact from inclusion bodies. This was then solubilized in guanidinium hydrochloride, immobilized on a cobalt-containing resin, and after dialysis against renaturation buffer, was quantitatively transformed to mature enzyme. However, when a mutation was introduced into the mature sequence to inactivate thermolysin, the proenzyme was not processed either in vivo or in vitro. In addition, mutated prothermolysin was not processed by exogenous thermolysin under a variety of experimental conditions. The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.
Marie-Claire
C
C
Departement 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.
Roques
B P
BP
Beaumont
A
A
D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D004789:Enzyme Activation / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D017510:Protein Folding; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013820:Thermolysin / Q000096:biosynthesis* / Q000378:metabolism
D011498:Protein Precursors; D011994:Recombinant Proteins; D013820:Thermolysin