E2 Conjugating - Antibodies
Activating enzymes are the initiators of the conjugation cascade for ubiquitin and ubiquitin-like modifiers. Antibodies to specific enzymes allow for the detection, location and depletion of these activities depending on your application.
These enzymes perform the second step in Ub (or UBL) conjugation reactions by forming a thiolester linkage between its active site cysteine with the C-terminal glycine of the modifier. E2 enzymes function alone and in conjunction with E3 ligase to catalyze the attachment of Ub (or UBL) proteins to acceptor lysine residues of target proteins to form isopeptide bonds. E2 enzymes act via selective protein–protein interactions with the E1 and E3 enzymes. It transfers the activated Ub/UBL to downstream effectors (E3s) to facilitate substrate modification. While E3s are involved in substrate selection, E2s are the main determinants for the lysine linkage present in Ub/UBL chains or substrates and thus directly influences the cellular fate of the substrate. The target lysine makes a nucleophilic attack on the carbonyl group of the Ub/Ubl-E2 thioester linkage upon deprotonation.
All E2s have in common a highly conserved 150–200 amino-acid (14-16kD) catalytic UBC domain which includes an active-site cysteine residue. This UBC domain is ~35% conserved among family members and substitution of the active site residue abolishes E2 activity. There are 3 main classes of E2 enzymes: Class I enzymes contain only the UBC domain. Class II and Class III enzymes have additional C- or N-terminal extensions respectively. These flanking regions are involved in E3 ligase interaction, substrate selection, dimerization and additional processes. The E2 enzymes that function in autophagy (Apg3 and Ap10) are not close in sequence to classical E2s, and there are also E2 variants proteins (UEVs) that lack the catalytic site cysteine and act by forming heterodimers with E2 containing functional active sites. There is also a dedicated E2 (Use1) that functions only with the alternate and newly identified E1 activating enzyme for Ub (UBE1L2, Uba6).
There are ~ 60 human E2s that function in conjunction with Ub, and related UBLs have 1 or 2 conjugating enzymes that are specific to those pathways. There are several E2s that function in ubiquitination, while UbcH9 is dedicated to SUMOylation, UbcH12 and Ube2F to