| Abstract
| - Ubiquitin-conjugating enzymes (E2s or Ubcs) are essential components in the ubiquitinationapparatus. These enzymes accept ubiquitin from an E1 enzyme and then, usually with the aid of an E3enzyme, donate the ubiquitin to the target protein. The function of E2 relies critically on the chemistry ofits active site cysteine residue since this residue must form a thioester bond with the carboxyl terminusof ubiquitin. Despite the plethora of structural information that is available, there has been a notabledearth of information regarding the chemical basis of E2 function. Toward filling this large void in ourunderstanding of E2 function, we have examined the pKa of the active site cysteine using a combinationof experimental and theoretical approaches. We find, remarkably, that the pKa of the active site cysteineresidue is elevated by approximately 2 pH units above that of a free cysteine. We have identified residuesthat contribute to the increase in this pKa. On the basis of experimental values obtained with three differentE2 proteins, we believe this to be a general and important characteristic of E2 protein chemistry. Sequencecomparison suggests that the electrostatic environment is maintained not through strict residue conservationbut through different combinations of residues near the active site. We propose that the elevated pKa isa regulatory mechanism that prevents the highly exposed cysteine residue in free E2 from reactingpromiscuously with electron deficient chemical moieties in the cell.
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