Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K)

Cecile M. Pickart, Margaret T. Haldeman, Eileen M. Kasperek, Zhijian Chen

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33 Scopus citations


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 (>20-fold reduction in k(cat)/K(m) 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(35K) 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.

Original languageEnglish (US)
Pages (from-to)14418-14423
Number of pages6
JournalJournal of Biological Chemistry
Issue number20
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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