title: Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans.
abstract: The contribution of lysine-128 within the active site of Anacystis nidulans d-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coli and the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by beta-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential epsilon-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.
authors.affiliation: Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Herts. AL5 2JQ, U.K.
authors.forename: G
authors.initials: G
authors.lastname: Bainbridge
authors.forename: P J
authors.initials: PJ
authors.lastname: Anralojc
authors.forename: P J
authors.initials: PJ
authors.lastname: Madgwick
authors.forename: J E
authors.initials: JE
authors.lastname: Pitts
authors.forename: M A
authors.initials: MA
authors.lastname: Parry
mesh_terms: D020134:Catalytic Domain; D000458:Cyanobacteria / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D009154:Mutation; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D010710:Phosphates; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine