![]() These small prolyl isomerases catalyse prolyl isomerizations much better in short unstructured oligopeptides ( Stein, 1993 Fischer, 1994) than in refolding protein chains ( Schmid et al., 1993). The prolyl isomerase function is thought to be important for protein folding, and initial experiments ( Stoller et al., 1995) showed that the trigger factor is much more effective as a folding catalyst than cyclophilin, FKBP or parvulin. Proteolytic fragments of trigger factor, which encompass the putative FKBP domain (residues 132–247 and 145–251, respectively) and a recombinant form of the 148–249 fragment retained the full prolyl isomerase activity of the intact protein, when assayed with proline-containing oligopeptides ( Hesterkamp and Bukau, 1996 Stoller et al., 1996). This homology is significant only for the residues that are necessary for substrate binding and activity. A weak sequence homology was noted between the 165–240 region of the trigger factor and human FKBP12 ( Callebaut and Mornon, 1995 Hesterkamp et al., 1996). It is, however, neither inhibited by FK 506 nor by cyclosporin A. In its prolyl isomerase activity the trigger factor resembles the FK 506 binding proteins (FKBPs). They discovered such an enzyme and identified it as the trigger factor. Both groups found crosslinking of presecretory and non-secretory proteins to the trigger factor.Īt the same time Fischer and co-workers ( Stoller et al., 1995) searched for a ribosome-bound prolyl isomerase in E.coli. The groups of Bukau ( Hesterkamp et al., 1996) and Luirink ( Valent et al., 1995) incorporated photoactivatable chemical crosslinkers into nascent proteins and, after arresting translation, searched for proteins which possibly bind to and are crosslinked with these newly formed protein chains, while they are still associated with the ribosome. Recently, the trigger factor was rediscovered by three groups, which used different experimental approaches to investigate early processes in cellular protein maturation. They found that the trigger factor interacted with the export-competent form of a precursor protein, proOmpA, and that it bound to the large subunit of the ribosome ( Crooke et al., 1988). It was discovered originally by Wickner and co-workers in 1987 when they searched for cytosolic components which are involved in the export of secretory proteins ( Crooke and Wickner, 1987 Lill et al., 1988). The trigger factor is an abundant soluble protein of Escherichia coli with a M r of 48 kDa. the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline-limited protein folding. Together, this suggests that the good substrate binding, i.e. Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain. The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800-fold reduced and no longer inhibited by an unfolded protein. An unfolded protein inhibits the trigger factor in a competitive fashion. In contrast, the catalytic constant k cat is small and shows a value of 1.3 s −1 at 15☌. The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low K m value of 0.7 μM. In its enzymatic mechanism the trigger factor follows the Michaelis–Menten equation. It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins. The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline-limited steps in protein folding with a very high efficiency.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |