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Volume 271, Number 43, Issue of October 25, 1996 pp. 26477-26481
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Roles of the Prosequence of Thermolysin in Enzyme Inhibition and Folding in Vitro

(Received for publication, June 21, 1996, and in revised form, August 5, 1996)

From the Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM, Unité de Recherche associée, D1500, CNRS, Université René Descartes, Unité de Formation de Recherche des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l'Observatoire, 75270 Paris Cedex 06, France

The zinc endopeptidase thermolysin (EC 3.4.24.27), an extracellular enzyme from Bacillus thermoproteolyticus, is synthesized as a preproprotein, with the prosequence (204 residues) being two-thirds the size of the mature enzyme (316 residues). This prosequence, expressed in and purified from Escherichia coli, inhibited thermolysin in vitro with an IC₅₀ value of 14 nM. It also inhibited a closely related enzyme produced by Bacillus stearothermophillus, albeit with a 16-fold higher IC₅₀ value (220 nM). The IC₅₀ value for thermolysin inhibition was also increased 15-fold (210 nm) by a monoclonal antibody that recognizes an epitope close to, but not forming a part of, the active site. At a prosequence concentration of 5 µM a mammalian, thermolysin-like enzyme, neutral endopeptidase 24.11, was not inhibited. The prosequence appeared to act as a mixed, noncompetitive inhibitor of thermolysin activity, with a K_i value of 6 nM for its interaction with the enzyme alone and a K_i value of 20 nM for its interaction with the enzyme-substrate complex. In addition, when thermolysin was denatured in 6 M guanidinium hydrochloride at acid pH and then brought to neutral pH by rapid dilution, the prosequence was found to facilitate the recovery of active enzyme in a stoichiometric manner.

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