Core domain mutation (S86Y) selectively inactivates polyubiquitin chain synthesis catalyzed by E2-25K

Biochemistry
L D MastrandreaC M Pickart

Abstract

The mammalian ubiquitin conjugating enzyme known as E2-25K catalyzes the synthesis of polyubiquitin chains linked exclusively through K48-G76 isopeptide bonds. The properties of truncated and chimeric forms of E2-25K suggest that the polyubiquitin chain synthesis activity of this E2 depends on specific interactions between its conserved 150-residue core domain and its unique 50-residue tail domain [Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537]. In the present study, we provide strong support for this model by showing that a point mutation in the core domain (S86Y) mimics the effect of deleting the entire tail domain: the ability to form an E2 approximately ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (>/=100-fold reduction in kcat/Km). The properties of E2-25K enzymes carrying the S86Y mutation indicate that this mutation strengthens the interaction between the core and tail domains: both free and ubiquitin-bound forms of S86Y-25K are completely resistant to tryptic cleavage at K164 in the tail domain, whereas wild-type enzyme is rapidly cleaved at this site. Other properties of S86Y-26K suggest that the active site of this mutant enzyme i...Continue Reading

References

Aug 9, 1996·The Journal of Biological Chemistry·M A KalchmanM R Hayden
Jan 1, 1996·Annual Review of Genetics·M Hochstrasser
Sep 20, 1997·The Journal of Biological Chemistry·J PiotrowskiC M Pickart
Apr 16, 1998·The Journal of Biological Chemistry·D K WorthylakeC P Hill

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Citations

Sep 10, 2010·The Journal of Biological Chemistry·Sunggeon KoWeontae Lee
Jul 27, 2006·Proceedings of the National Academy of Sciences of the United States of America·Dennis FliermanVincent Chau
Aug 23, 2011·Nature Structural & Molecular Biology·Anna PlechanovováRonald T Hay
Nov 3, 2004·Trends in Molecular Medicine·Sungmin Song, Yong-Keun Jung
Aug 13, 2013·Biochimica Et Biophysica Acta·Yoonhee BaeSeongman Kang
Oct 7, 2003·Molecular Cell·Sungmin SongYong-Keun Jung
Jul 25, 2000·The Journal of Biological Chemistry·T Yao, R E Cohen
Oct 24, 2007·The Journal of Biological Chemistry·J Torin HuzilMichael J Ellison

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