A conserved catalytic residue in the ubiquitin-conjugating enzyme family

Author:

Wu, P. Y.

Hanlon, M.

Eddins, M.

Tsui, C.

Rogers, R. S.

Jensen, J. P.

Matunis, M. J.

Weissman, A. M.

Wolberger, C. P.

Pickart, C. M.
Author Address

Johns Hopkins Univ, Bloomberg Sch Publ Hlth, Dept Biochem & Mol Biol, 615 N Wolfe St, Baltimore, MD 21205 USA Johns Hopkins Univ, Bloomberg Sch Publ Hlth, Dept Biochem & Mol Biol, Baltimore, MD 21205 USA Johns Hopkins Univ, Dept Biophys & Biophys Chem, Program Mol Biophys, Baltimore, MD 21205 USA Johns Hopkins Univ, Sch Med, Howard Hughes Med Inst, Baltimore, MD 21205 USA NCI, Regulat Prot funct Lab, Ctr Canc Res, NIH, Frederick, MD 21702 USA Pickart CM Johns Hopkins Univ, Bloomberg Sch Publ Hlth, Dept Biochem & Mol Biol, 615 N Wolfe St, Baltimore, MD 21205 USA

Abstract:

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.

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