Biochemical and biophysical characterization of compartmentalizing proteases from the hyperthermophilic microorganism Pyrococcus furiosus.

摘要

The focus of this study was on the biochemical and biophysical characteristics of two multi-subunit compartmentalizing proteases from the hyperthermophilic archaeon Pyrococcus furiosus (Topt = 100°C). The first protease was an oligopeptidase, PfpI (P&barbelow;yrococcus f&barbelow;uriosus p&barbelow;rotease I&barbelow;), and the second was a proteinase, called the proteasome. Both proteases are ubiquitous in all domains of life. However, they are theorized to have distinctly different roles within P. furiosus. The proteasome may be one of the primary proteinases, with access to its active sites tightly controlled by ATPase regulators that appear to be dependent on cellular environment. In contrast, the role of PfpI may be degradation of the smaller peptides that result from proteasome and other proteinase action.;PfpI is a homo-multimer of 18.8-kDa subunits that assemble into hexameric rings. These rings then stack to form dodocamers and higher forms, with three active sites buried in hindered positions within each ring. Trimer, hexamer, and dodecamer forms were purified separately, with the dodecamer at least three-fold more specifically active than the smaller forms.;The 20S proteasome, along with a theorized ATP-dependent regulator PAN (proteasome-activating nucleotidase), was investigated from several angles. Both enzymes, including native and recombinant forms, were tested for biochemical and biophysical characteristics as isolated structures and in combination. In particular, the PAN ATPase activity was tested primarily to observe its effects on different forms of the proteasome.;Distinct differences in activity, stability, and level of ATPase-based stimulation were observed for the various proteasome forms. These differences were based on the presence or absence of one of the three subunits and the assembly temperature. The beta-2 subunit appeared to be the catalytic center for proteinase activity, while the beta-I subunit played a stabilizing role. PAN was able to stimulate the native form of the proteasome during degradation of polypeptides but inhibited the native heat-shocked form in the same reactions. It was concluded that PAN, which is highly up-regulated during heat shock, may stimulate the native proteasome form, while the heat-shocked proteasome (containing higher levels of beta-1) may associate with a different set of regulating proteins. (Abstract shortened by UMI.)