HslVUATP-dependent Protease UtilizesMaximally Six among Twelve Threonine Active Sites during Proteolysis*□S

HslVU is a bacterial ATP-dependent protease distantly re-lated to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecamericHslV protease. As a homolog of the 20 S proteasome -subunits, HslV also uses the N-terminal threo-nine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activ-ity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only6 active sites is sufficient to sup-port full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activ-ity. Using proteasome inhibitors, we also demonstrate that sub-strate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites i