Function of Lys-166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase as examined by site-directed mutagenesis.
Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman, F.C., Milanez, S., and Lee, E.H. (1985) J. Biol. Chem. 260, 13968-13975). In attempts to clarify the role of Lys-166 of the carboxylase, we have used site-directed mutagenesis to replace this lysyl residue with glycine, alanine, serine, glutamine, arginine, cysteine, or histidine. All seven of these mutant proteins, purified by immunoaffinity chromatography, are severely deficient in carboxylase activity; the serine mutant, which is the most active, has a kcat only 0.2% that of the wild-type enzyme. Although low, the carboxylase activity displayed by some of the mutant proteins proves that Lys-166 is not required for substrate binding and argues that the detrimental effects brought about by amino acid substitutions at position 166 do not reflect gross conformational changes. As demonstrated by their ability to tightly bind a transition-state analogue (2-carboxyarabinitol 1,5-bisphosphate) in the presence of CO2 and Mg2+, some of the mutant proteins undergo the carbamylation reaction that is required for activation of the wild-type enzyme. Since Lys-166 is required neither for activation (i.e. carbamylation by CO2) nor for substrate binding, it must be essential to catalysis. When viewed within the context of previous related studies, the results of site-directed mutagenesis are entirely consistent with Lys-166 functioning as the base that initiates catalysis by abstracting the C-3 proton from ribulosebisphosphate. An alternative possibility that Lys-166 acts to stabilize a transition state in the reaction pathway cannot be rigorously excluded.
D000595:Amino Acid Sequence; D001483:Base Sequence; D007700:Kinetics; D008239:Lysine; D009154:Mutation; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*
D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine