Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex.
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.
Tatham
Michael H
MH
Centre for Biomolecular Sciences, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, Scotland.
Chen
Yuan
Y
Hay
Ronald T
RT
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004952:Esters; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D025842:SUMO-1 Protein / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D017931:DNA Primers; D018076:DNA, Complementary; D004952:Esters; D025842:SUMO-1 Protein; D001224:Aspartic Acid; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine