The 2OS proteasome from Thermoplasma acidophilum: Insight on the catalytic mechanism for scission in a barrel

The proteolytic activities of the proteasome multienzyme complex are essential to cell life. The archeabacterium Thermoplasma Acidophilum 20S proteasome has been selected for mechanistic studies based on its structural simplicity, exhibiting a homodimeric subunit array with an alpha 14beta14 barrel-shaped oligomeric structure versus the similar but more diverse population of subunits found in the eukaryotic proteasome.; In studies designed to provide insight into how substrate proteins gain entry into the proteasome complex, we have examined the interactive behavior of the proteasome with horse heart ferricytochrome c (cyt c). The chromophoric heme and tryptophan moieties within cyt c serve as useful probes to the presence and intactness of the substrate. Under conditions of temperature and denaturant which cause cyt c to unfold but do not alter the native state of the thermophilic proteasome, we have demonstrated that cyt c can gain entrance into the constricted entrance of the proteasome and is available for proteolytic degradation. Furthermore, in the presence of inhibitors of the proteosome we have shown that the unfolded cyt c can be sequestered inside the proteasome complex to form a quasi-stable host-guest complex in which the cyt c appears to be in a near native-like conformation.; Therefore, this system provides the ability to monitor a variety of things, including: (1) the degradative behavior of the proteasome on cyt c without the inhibitor and (2) the sequestering of cyt c as an intact protein inside the central chamber of the inhibited proteasome.; Data collected from UV-visible spectroscopy, high performance liquid chromatography (HPLC) and mass spectrometry provide evidence that intact cyt c can be sequestered within the inhibited proteasome. HPLC data show that accumulation of cyt c within the proteasome reaches saturation at an occupancy of one cyt c molecule per proteasome molecule with inhibition, and without inhibition substrate turnover can be observed.; In digest studies using the proteasome and horse heart cyt c, examination of peptide fragments reveals the effect of the covalently bound heme group on the proteasome's ability to digest cyt c. HPLC data show that the N-terminal region of cyt c is selectively cleaved into large heme bearing fragments that can be subsequently cleaved into shorter fragments. Both heme and non-heme bearing fragment amino acid sequences have been identified by MALDI mass spectrometry.