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• Ubiquitin-activating enzyme(E1s): This requires ATP and involves the initial
  formation of a ubiquitin–adenylate intermediate. Ubiquitin is then transferred to a
  cysteine residue in the active site of the E1 with the concomitant release of AMP.


• Ubiquitin-conjugating enzymes(E2s): These receive the ubiquitin from the E1 by a
  transthioesterification reaction. 20–30 different E2s are known.


• Ubiquitin protein ligases(E3s): These interacts with both the E2 and the substrate
  by means of either a HECT or RING binding domain on the E3 resulting in the
  transfer of the ubiquitin to a lysine residue in the substrate. Results from the Human
  Genome Project provide evidence for the existence of several hundred of these
  E3 enzymes.


• The process either stops after monoubiquitination or is repeated resulting in the
  attachment of four or more ubiquitin residues to the substrate, a process called
  polyubiquitination.

E1 E2 E2

(b)

Ubiquitin-

activating

enzyme

Ubiquitin

Peptides

Ubiquitin-carrier

protein; Ubiquitin-

conjugating

enzyme, UBC

S

E3

E2

S

19S

20S 26S Proteasome

19S

S

Peptides

E3

S

S

S

RING finger-

containing

E3s

substrates

Proteolytic

HECT

domain-

containing

E3s

(a)

E3

E3

Fig.15.15The ubiquitin–proteasome system (UPS). Ubiquitin is first activated to a high-energy intermediate

by E1. It is then transferred to a member of the E2 family of enzymes. From E2 it can be transferred directly

to the substrate (S) that is bound specifically to a member of the ubiquitin ligase family of proteins, E3 (a).

This occurs when the E3 belongs to the RING finger family of ligases. In the case of a HECT-domain-containing

ligase (b), the activated ubiquitin is transferred first to the E3 before it is conjugated to the E3-bound substrate.

Additional ubiquitin moieties are added successively to the previously conjugated moiety to generate a

polyubiquitin chain. The polyubiquitinated substrate binds to the 26S proteasome complex, the substrate is

degraded to short peptides, and free and reusable ubiquitin is released through the activity of deubiquitinating

enzymes (DUBs). (Reproduced from A. Ciechanover and R. Ben-Saadon (2004). N-terminal ubiquitination:

more protein substrates join in.Trends in Cell Biology, 14 , 103–106, by permission of Elsevier Science.)

622 Enzymes