Previously we have examined the inactivation of unpurified extracellular
proteinase from Pseudomonas fluorescens 22F diluted in demineralized
(Schokker & van Boekel, 1998) in the range 40–70°C. It appeared
inactivation was most probably caused by intermolecular autoproteolysis,
the hydrolysis of unfolded proteinase molecules by native (not yet unfolded)
molecules. It has been reported that purification of proteinases from Pseudomonas
spp. enhances the susceptibility of the proteinase to autoproteolysis (Barach
1976; Griffiths et al. 1981; Leinmüller &
Christophersen, 1982; Kroll, 1989; Kumura
et al. 1991). On the other hand, when the proteinase is heated
in milk or when
proteins are added to the enzyme solution, the rate of inactivation by
diminishes (Barach et al. 1978; Kroll & Klostermeyer,
1984; Stepaniak et al. 1991).
Apparently, proteins stabilize the proteinase against inactivation by autoproteolysis.
Substrate or other ligands stabilize many enzymes against limited proteolysis.
Binding of these substances to the enzyme molecule, either to the catalytic
to amino acid residues on the enzyme molecule surface, may impose steric
so that the susceptible peptide bonds are protected against proteolysis
1978). Such binding may also cause a conformational change of the enzyme
such that susceptible peptide bonds cannot be attacked or that the conformation
stabilized against unfolding (Mihalyi, 1978). In the latter case an increase
denaturation temperature (Td) would be expected.
In the case of proteinases, addition of substrate to the enzyme solution
protect the enzyme by a third mechanism. Besides autoproteolysis of the
the added proteins can be digested. An enzyme molecule digesting a protein
available at the same time for autoproteolysis, so that the substrate may
act as a
competitive inhibitor against autoproteolysis.
The aim of this study was to determine the mechanism of protection of
proteinase from Ps. fluorescens 22F by sodium caseinate.