Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein.
It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to 125I-lysozyme, the conjugates produced are very tightly bound to E3, as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein. In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2, a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2.E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.
Reiss
Y
Y
Unit of Biochemistry, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
Heller
H
H
Hershko
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004151:Dipeptides / Q000494:pharmacology; D007700:Kinetics; D007782:Lactoglobulins / Q000378:metabolism; D008025:Ligases / Q000097:blood* / Q000302:isolation & purification; D009113:Muramidase / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D044767:Ubiquitin-Protein Ligases
D004151:Dipeptides; D007782:Lactoglobulins; D000255:Adenosine Triphosphate; D044767:Ubiquitin-Protein Ligases; D009113:Muramidase; D008025:Ligases