The activation kinetics of constitutive and IFNγ‐stimulated 20S proteasomes obtained with homomeric (recPA28α, recPA28β) and heteromeric (recPA28αβ) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling.

The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28α/RecP28β/RecP28α+ RecP28β) exhibit biphasic characteristics which can be attributed to a low‐level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28αβ complex is about two orders of magnitude higher than towards the homomeric PA28α and PA28β complexes.

Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin‐like, trypsin‐like and peptidylglutamyl‐peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.