22 other proteins in multiple species, including ubiquitin itself. There is also increasing evidence for nonlysine residues as ubiquitylation targets using non-amine groups, such as the sulfhydryl group on cysteine, and the hydroxyl group on threonine and serine. The end result of this process is the addition of one ubiquitin molecule (monoubiquitylation) or a chain of ubiquitin molecules (polyubiquitination) to the substrate protein. Ubiquitination requires three types of enzyme: ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, known as E1s, E2s, and E3s, respectively. The process consists of three main steps: Activation: Ubiquitin is activated in a two-step reaction by an E1 ubiquitin-activating enzyme, which is dependent on ATP. The initial step involves production of a ubiquitin-adenylate intermediate. The E1 binds both ATP and ubiquitin and catalyses the acyl-adenylation of the C-terminus of the ubiquitin molecule. The second step transfers ubiquitin to an active site cysteine residue, with release of AMP. This step results in a thioester linkage between the C-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group. The human genome contains two genes that produce enzymes capable of activating ubiquitin: UBA1 and UBA6. Conjugation: E2 ubiquitin-conjugating enzymes catalyse the transfer of ubiquitin from E1 to the active site cysteine of the E2 via a trans(thio)esterification reaction. In order to perform this reaction, the E2 binds to both activated ubiquitin and the E1 enzyme. Humans possess 35 different E2 enzymes, whereas other eukaryotic organisms have between 16 and 35. They are characterised by their highly conserved structure, known as the ubiquitin-conjugating catalytic (UBC) fold. Ligation: E3 ubiquitin ligases catalyse the final step of the ubiquitination cascade. Most commonly, they create an isopeptide bond between a lysine of the target protein and the C-terminal glycine of ubiquitin. In general, this step requires the activity of one of the hundreds of E3s. E3 enzymes function as the substrate recognition modules of the system and are capable of interaction with both E2 and substrate. Some E3 enzymes also activate the E2 enzymes. E3 enzymes possess one of two domains: the homologous to the E6-AP carboxyl terminus (HECT) domain and the really interesting new gene (RING) domain (or the closely related U-box domain). HECT domain E3s transiently bind ubiquitin in this process (an obligate thioester intermediate is formed with the active-site cysteine of the E3), whereas RING domain E3s catalyse the direct transfer from the E2 enzyme to the substrate. The anaphase-promoting