THE ROLES OF THE PROSEQUENCE OF THERMOLYSIN IN ENZYME-INHIBITION AND FOLDING IN-VITRO

The zinc endopeptidase thermolysin (EC 3.4.24.27), an extracellular en zyme from Bacillus thermoproteolyticus, is synthesized as a preproprot ein, with the prosequence (204 residues) being two thirds the size of the mature enzyme (316 residues). This prosequence, expressed in and p urified from Escherichia coil, inhibited thermolysin in vitro with an IC50 value of 14 nM. It also inhibited a closely related enzyme produc ed by Bacillus stearothermophillus, albeit with a 16-fold higher IC50 value (220 nM). The IC50 value for thermolysin inhibition was also inc reased 15-fold (210 nm) by a monoclonal antibody that recognizes an ep itope close to, but not forming a part of, the active site. At a prose quence concentration of 5 mu M a mammalian, thermolysin-like enzyme, n eutral endopeptidase 24.11, was not inhibited. The prosequence appeare d 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 an d a K-i' value of 20 nM for its interaction with the enzyme substrate complex. In addition, when thermolysin was denatured in 6 M guanidiniu m hydrochloride at acid pH and then brought to neutral pH by rapid dil ution, the prosequence was found to facilitate the recovery of active enzyme in a stoichiometric manner.