Structure and function of ubiquitin: evidence for differential interactions of arginine-74 with the activating enzyme and the proteases of ATP-dependent proteolysis.
Ubiquitin was modified with the anionic, arginine-specific reagent 4-(oxoacetyl)phenoxyacetic acid in order to study the relationship between structure and function of the molecule. Four different derivatives (A, B, C, and D) were purified from the reaction mixture by anion-exchange high-performance liquid chromatography and subjected to tryptic peptide mapping to determine the location of the modification(s). These derivatives were stable throughout the procedures required for purification, tryptic hydrolysis, and peptide mapping. Derivative A was modified at arginine-42, derivative B at arginine-72, derivative C at arginines-42 and -72, and derivative D at arginine-74. Modification of ubiquitin with 14C-labeled 4-(oxoacetyl)phenoxyacetic acid indicated that the reagent formed a stable, 1:1 complex with arginine residues of the protein. Native ubiquitin and each of the four derivatives were tested for their ability to stimulate 32P exchange between ATP and pyrophosphate, a reaction catalyzed by enzyme 1 of the ubiquitin-dependent proteolytic pathway. A and C were capable of promoting this exchange at a rate only 15% that of native ubiquitin, B stimulated the exchange to 25%, and D stimulated exchange to 60% of the native level. None of the derivatives was capable of promoting a significant level of ubiquitin-dependent proteolysis. D was capable of forming conjugates with exogenous and endogenous proteins to an extent very similar to that of native ubiquitin, suggesting that its inability to stimulate ubiquitin-dependent proteolysis was due to a defect in a step beyond that of conjugate formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Duerksen-Hughes
P J
PJ
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Xu
X X
XX
Wilkinson
K D
KD
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001120:Arginine; D002417:Cattle; D004912:Erythrocytes / Q000378:metabolism; D006016:Glycolates / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism*; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin; D014452:Ubiquitins / Q000097:blood*
D000596:Amino Acids; D006016:Glycolates; D010446:Peptide Fragments; D014452:Ubiquitins; C052188:4-(oxoacetyl)phenoxyacetic acid; D000255:Adenosine Triphosphate; D001120:Arginine; D010447:Peptide Hydrolases; D014357:Trypsin