INTRAMOLECULAR PROCESSING OF PROTHERMOLYSIN

Thermolysin, an extracellular zinc endopeptidase from Bacillus thermop roteolyticus, is synthesized as a pre-proenzyme and the prosequence ha s been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O'Donohue, M. J., and Beaumont, A. (19 96) J. Biol. Chem. 271, 26477-26481), To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether i t is an intermolecular or intramolecular process, N-terminal histidine -tagged prothermolysin was expressed in Escherichia coil, Although par tial processing to mature enzyme occurred, most of the proenzyme was r ecovered intact from inclusion bodies, This was then solubilized in gu anidinium hydrochloride, immobilized on a cobalt-containing resin, and after dialysis against renaturation buffer, was quantitatively transf ormed to mature enzyme, However, when a mutation was introduced into t he mature sequence to inactivate thermolysin, the proenzyme was not pr ocessed either in vivo or in vitro. In addition, mutated prothermolysi n was not processed by exogenous thermolysin under a variety of experi mental conditions, The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.