Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K).
Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. Ubiquitin-conjugating enzyme E2(25K) utilizes isolated ubiquitin as the substrate for synthesis of such chains, in which successive ubiquitin units are linked by isopeptide bonds involving the side chain of Lys-48 of one ubiquitin and the COOH group of Gly-76 of the next. During continuous synthesis of multiubiquitin chains in the presence of purified ubiquitin-activating enzyme and E2(25K), there was a slight discrimination against radioiodinated ubiquitin (2.3-fold reduction in specific radioactivity of diubiquitin relative to value expected for no discrimination). Single-turnover experiments employing stoichiometrically iodinated ubiquitin derivatives indicated that E2(25K) discriminates extremely strongly (greater than 20-fold reduction in kcat/Km for diubiquitin synthesis) against ubiquitin that is monoiodinated at Tyr-59. The modest overall selection effect observed in continuous reactions is in part due to the occurrence of discrimination only when iodotyrosylubiquitin is the acceptor (Lys-48 donor) in diubiquitin synthesis; iodotyrosylubiquitin is kinetically competent when it is the species being transferred to native ubiquitin. The competence as acceptor of a site-directed mutant form of ubiquitin bearing a Tyr to Phe substitution at position 59 indicated that discrimination against iodotyrosylubiquitin by E2(25K) is not due to loss of the hydrogen-bonding interactions of Tyr-59. Rather, iodotyrosylubiquitin may be unable to react with the ubiquitin adduct of E2(25K) for steric reasons. Discrimination against iodotyrosylubiquitin as acceptor is unique to E2(25K) among three enzymes surveyed: iodotyrosylubiquitin is a fully competent acceptor in diubiquitin synthesis catalyzed by E2(25K) and is also utilized for multiubiquitin chain synthesis by E2(14K) and ubiquitin-protein ligase. These findings should assist in the design of future studies concerning E2(25K) structure and function.
Pickart
C M
CM
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
Haldeman
M T
MT
Kasperek
E M
EM
Chen
Z
Z
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014452:Ubiquitins; D014443:Tyrosine; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases