Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction.
Diverse approaches that include site-directed mutagenesis have indicated a catalytic role of Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To determine whether Lys-329 is required for the initial enolization of ribulose bisphosphate or for some subsequent step in the overall reaction pathway, the competence of position 329 mutant proteins (devoid of carboxylase activity) in catalyzing exchange of solvent protons with the C-3 proton of substrate has now been examined. Irrespective of the amino acid substitution for Lys-329, the mutant protein retains 2-6% of the wild-type activity in the proton exchange reaction. The complete stability of ribulose bisphosphate during the enolization catalyzed by mutant protein suggests that the major effect of Lys-329 is to facilitate the addition of gaseous substrates (CO2 or O2) to the enediol intermediate. The exchange reaction requires Mg2+, is CO2-dependent, and is inhibited by the transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate. A mutant protein in which Lys-191, the site for carbamylation by CO2 in an obligatory activation step, is replaced by a cysteinyl residue totally lacks proton exchange activity. Barely detectable exchange activity (approximately 0.2% of wild-type) is displayed by the Lys-166----Cys mutant protein, consistent with the previously implicated role of Lys-166 in the deprotonation of ribulose bisphosphate. Retention of exchange activity by the Glu-48----Gln mutant protein, which is slightly active in overall carboxylation, demonstrates that active site Glu-48, like Lys-329, exerts its major effect at some step subsequent to the initial enolization.
Hartman
F C
FC
Biology Division, Oak Ridge National Laboratory, Tennessee 37831.
Lee
E H
EH
D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D008239:Lysine / Q000378:metabolism*; D009154:Mutation; D011522:Protons; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*
D011522:Protons; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine