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 degradat ion of ubiquitin-protein conjugates. Ubiquitin (Ub)-activating enzyme (E1) catalyzes the first, MgATP-dependent step in the conjugative reac tion sequence. With wild type ubiquitin, the product of the E1 reactio n is a ternary complex (E1-Ub.AMP-Ub) containing one thiol-linked ubiq uitin (via the Uh COOH terminus, Gly-76) and one tightly bound ubiquit in adenylate. The thiol-linked ubiquitin is subsequently transferred t o the thiol of a ubiquitin-conjugating enzyme (E2 protein); the latter adduct is the proximal donor of ubiquitin to the target protein. A mu tant ubiquitin, bearing a Gly to Ala substitution at the COOH terminus (G76A-ubiquitin), was characterized as a substrate for E1. G76A-ubiqu itin 1) supported PP1-ATP exchange poorly (500-fold decrease in k(cat) /K-m); 2) did not produce detectable AMP-Ub with native E1; 3) produce d stoichiometric AMP-Ub with thiol-blocked E1; 4) gave a stoichiometri c burst of ATP consumption (1 mol/mol E1) with either native or thiol- blocked E1; 5) supported E1-ubiquitin thiol ester formation with nativ e E1; 6) supported several downstream reactions of the proteolytic pat hway at similar to 20% of the rate of wild type ubiquitin. These resul ts indicate that G76A-ubiquitin gives a binary E1 thiol ester complex with native E1, due to the failure of the E1-ubiquitin thiol eater 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, occup ancy of the nucleotide/adenylate site of E1, by either MgATP or AMP-Ub , was found to stimulate ubiquitin transthiolation between E1 and E2 p roteins. The intermediacy of ubiquitin adenylate thus provides a previ ously unrecognized catalytic advantage in the E1 mechanism.