Determination of the pKa of the N-terminal amino group of ubiquitin by NMR

Sci Rep. 2017 Mar 2;7:43748. doi: 10.1038/srep43748.


Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pKa of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Protein Conformation
  • Protein Interaction Domains and Motifs*
  • Ubiquitin / chemistry*
  • Ubiquitin / metabolism
  • Ubiquitination


  • Ubiquitin