title: Pleiotropic effects of ATP.Mg2+ binding in the catalytic cycle of ubiquitin-activating enzyme.
abstract: Conjugation of ubiquitin and other Class 1 ubiquitin-like polypeptides to specific protein targets serves diverse regulatory functions in eukaryotes. The obligatory first step of conjugation requires ATP-coupled activation of the ubiquitin-like protein by members of a superfamily of evolutionarily related enzymes. Kinetic and equilibrium studies of the human ubiquitin-activating enzyme (HsUba1a) reveal that mutations within the ATP.Mg(2+) binding site have remarkably pleiotropic effects on the catalytic phenotype of the enzyme. Mutation of Asp(576) or Lys(528) results in dramatically impaired binding affinities for ATP.Mg(2+), a shift from ordered to random addition in co-substrate binding, and a significantly reduced rate of ternary complex formation that shifts the rate-limiting step to ubiquitin adenylate formation. Mutations at neither position affect the affinity of HsUbc2b binding; however, differences in k(cat) values determined from ternary complex formation versus HsUbc2b transthiolation suggest that binding of the E2 enhances the rate of bound ubiquitin adenylate formation. These results confirm that Asp(576) and Lys(528) are important for ATP.Mg(2+) binding but are essential catalytic groups for ubiquitin adenylate transition state stabilization. The latter mechanistic effect explicates the observed loss-of-function phenotype associated with mutation of residues paralogous to Asp(576) within the activating enzymes for other ubiquitin-like proteins.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Tokgöz
authors.forename: Richard N
authors.initials: RN
authors.lastname: Bohnsack
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Haas
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D010641:Phenotype; D017354:Point Mutation; D025801:Ubiquitin / Q000737:chemistry; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry*
chemical_list: D025801:Ubiquitin; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D044764:Ubiquitin-Activating Enzymes; D008274:Magnesium