The small ubiquitin-like
modifier SUMO-1 is covalently attached to lysine residues on target
proteins by a specific conjugation pathway involving the E1 enzyme
SAE1/SAE2 and the E2 enzyme Ubc9. In an ATP-dependent manner, the
C-terminus of SUMO-1 forms consecutive thiolester bonds with cysteine
residues in the SAE2 subunit and Ubc9, before the Ubc9.SUMO-1 thiolester
complex catalyzes the formation of an isopeptide bond between SUMO-1 and
the epsilon-amino group of the target lysine residue on the protein
substrate. The SUMO-1 conjugation pathway bears many similarities with
that of ubiquitin and other ubiquitin-like protein modifiers (Ubls), and
because of its production of a singly conjugated substrate and the lack of
absolute requirement in vitro for E3 enzymes, the SUMO-1/Ubc9 system is a
good model for the analysis of protein conjugation pathways that share
this basic chemistry. Here we describe methods of both steady-state and
half-reaction kinetic analysis of Ubc9, and use these techniques to
determine the role of two residues, Asp(100) and
Lys(101) of Ubc9 which are not found in E2 enzymes
from other protein conjugation pathways. These residues are found close to
the active site Cys in the tertiary structure of Ubc9, and although they
are shown to inhibit the transesterification reaction from SAE1/SAE2, they
are important for substrate recognition in the context of the thiolester
complex with SUMO-1.
PMID: 12641448 [PubMed -
indexed for MEDLINE]
