Substrate properties of site-specific mutant ubiquitin protein (G76A) reveal unexpected mechanistic features of ubiquitin-activating enzyme (E1).
Ubiquitin-mediated proteolysis proceeds via the formation and degradation of ubiquitin-protein conjugates. Ubiquitin (Ub)-activating enzyme (E1) catalyzes the first, MgATP-dependent step in the conjugative reaction sequence. With wild type ubiquitin, the product of the E1 reaction is a ternary complex (E1-Ub-AMP-Ub) containing one thiol-linked ubiquitin (via the Ub COOH terminus, Gly-76) and one tightly bound ubiquitin adenylate. The thiol-linked ubiquitin is subsequently transferred to the thiol of a ubiquitin-conjugating enzyme (E2 protein); the latter adduct is the proximal donor of ubiquitin to the target protein. A mutant ubiquitin, bearing a Gly to Ala substitution at the COOH terminus (G76A-ubiquitin), was characterized as a substrate for E1. G76A-ubiquitin 1) supported PPi-ATP exchange poorly (500-fold decrease in kcat/K(m); 2) did not produce detectable AMP-Ub with native E1; 3) produced stoichiometric AMP-Ub with thiol-blocked E1; 4) gave a stoichiometric burst of ATP consumption (1 mol/mol E1) with either native or thiol-blocked E1; 5) supported E1-ubiquitin thiol ester formation with native E1; 6) supported several downstream reactions of the proteolytic pathway at approximately 20% of the rate of wild type ubiquitin. These results indicate that G76A-ubiquitin gives a binary E1 thiol ester complex with native E1, due to the failure of the E1-ubiquitin thiol ester to undergo another round of adenylate synthesis; thus AMP-Ub is detected only if adenylate to thiol transfer is prevented by alkylating E1. The inability of G76A-ubiquitin to support ternary complex formation has implications for E1 active site structure. In other experiments, occupancy of the nucleotide/adenylate site of E1, by either MgATP or AMP-Ub, was found to stimulate ubiquitin transthiolation between E1 and E2 proteins. The intermediacy of ubiquitin adenylate thus provides a previously unrecognized catalytic advantage in the E1 mechanism.
Pickart
C M
CM
Department of Biochemistry, State University of New York, Buffalo 14214.
Kasperek
E M
EM
Beal
R
R
Kim
A
A
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002417:Cattle; D005998:Glycine / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
D009711:Nucleotides; D013438:Sulfhydryl Compounds; D014452:Ubiquitins; D000249:Adenosine Monophosphate; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes; D005998:Glycine