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 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 microM 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 Ki value of 6 nM for its interaction with the enzyme alone and a Ki' 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.
O'Donohue
M J
MJ
Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM, Paris, France.
Beaumont
A
A
D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013820:Thermolysin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D011498:Protein Precursors; D011994:Recombinant Proteins; D013820:Thermolysin