Ubiquitin and Ubl Cascade Enzymes (E1s, E2s and E3s)

The complexity of the ubiquitin and ubiquitin-like protein cascades is considerable. In mammals, there are some ten activating enzymes (E1s) known, some twenty plus conjugating enzymes (E2s), over eight hundred ligases (E3s), and approaching one hundred deconjugating enzymes. These varied components work in a hierarchical context and, for appropriate modification with ubiquitin or a Ubl to occur, the correct combination of E1, E2, E3, substrate, and deconjugating enzyme must all work in concert. The cascades for the ubiquitin-like proteins appear not to be as complex as that of ubiquitin with a reduced number of component possibilities.

The conjugation of ubiquitin and Ubls to substrates usually involves three steps: (i) an initial activation step catalyzed by a specific activating enzyme (E1) in which the C-terminus of the protein is activated for subsequent reaction; (ii) an intermediate step involving transfer of the protein from the E1 to a covalent linkage with a conjugating enzyme (E2); and (iii) in which the protein is transferred to an amino group on the substrate protein, is usually facilitated by a ligase enzyme (E3). The E2/E3 interaction determines the target of the protein, dictating its specific biological function. The availability of high purity/ high activity recombinant enzymes allows in vitro reconstitution of many of these pathway steps.