View the Current Issue

Article

  • The EMBO Journal (2003) 22, 5241 - 5250
  • doi:10.1093/emboj/cdg501

A conserved catalytic residue in the ubiquitin-conjugating enzyme family

Pei-Ying Wu1,2, Mary Hanlon3, Michael Eddins2, Colleen Tsui1, Richard S. Rogers1, Jane P. Jensen3, Michael J. Matunis1, Allan M. Weissman3, Cynthia P. Wolberger2,4 and Cecile M. Pickart1

  1. Department of Biochemistry and Molecular Biology/Bloomberg School of Public Health, Frederick, MD 21702, USA
  2. Program in Molecular Biophysics, Department of Biophysics and Biophysical Chemistry, Frederick, MD 21702, USA
  3. Regulation of Protein Function Laboratory, Center for Cancer Research, NCI/NIH, Frederick, MD 21702, USA
  4. Howard Hughes Medical Institute/School of Medicine, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA

Correspondence to:

Cecile M. Pickart, E-mail: cpickart@jhmi.edu

Received 16 June 2003; Accepted 12 August 2003; Revised 11 August 2003

Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In constrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.

  • Keywords:

    • catalytic mechanism,
    • E2,
    • E3,
    • isopeptide,
    • ubiquitin