title: The active site cysteine of ubiquitin-conjugating enzymes has a significantly elevated pKa: functional implications.
abstract: Ubiquitin-conjugating enzymes (E2s or Ubcs) are essential components in the ubiquitination apparatus. These enzymes accept ubiquitin from an E1 enzyme and then, usually with the aid of an E3 enzyme, donate the ubiquitin to the target protein. The function of E2 relies critically on the chemistry of its active site cysteine residue since this residue must form a thioester bond with the carboxyl terminus of ubiquitin. Despite the plethora of structural information that is available, there has been a notable dearth of information regarding the chemical basis of E2 function. Toward filling this large void in our understanding of E2 function, we have examined the pK(a) of the active site cysteine using a combination of experimental and theoretical approaches. We find, remarkably, that the pK(a) of the active site cysteine residue is elevated by approximately 2 pH units above that of a free cysteine. We have identified residues that contribute to the increase in this pK(a). On the basis of experimental values obtained with three different E2 proteins, we believe this to be a general and important characteristic of E2 protein chemistry. Sequence comparison suggests that the electrostatic environment is maintained not through strict residue conservation but through different combinations of residues near the active site. We propose that the elevated pK(a) is a regulatory mechanism that prevents the highly exposed cysteine residue in free E2 from reacting promiscuously with electron deficient chemical moieties in the cell.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: Blanton S
authors.initials: BS
authors.lastname: Tolbert
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Tajc
authors.forename: Helen
authors.initials: H
authors.lastname: Webb
authors.forename: Jessica
authors.initials: J
authors.lastname: Snyder
authors.forename: Jens E
authors.initials: JE
authors.lastname: Nielsen
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Miller
authors.forename: Ravi
authors.initials: R
authors.lastname: Basavappa
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002151:Calorimetry; D003545:Cysteine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D044763:Ubiquitin-Conjugating Enzymes; D003545:Cysteine