The active site cysteine of ubiquitin-conjugating enzymes has a significantly elevated pKa: functional implications.

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 pKa of the active site cysteine using a combination of experimental and theoretical approaches. We find, remarkably, that the pKa 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 pKa. On the basis of experimental values obtaine...