Ribulose 1,5-bisphosphate carboxylase. Effect on the catalytic properties of changing methionine-330 to leucine in the Rhodospirillum rubrum enzyme.
Oligonucleotide-directed mutagenesis of cloned Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase with a synthetic 13mer oligonucleotide primer was used to effect a change at Met-330 to Leu-330. The resultant enzyme was kinetically examined in some detail and the following changes were found. The Km(CO2) increased from 0.16 to 2.35 mM, the Km(ribulose bisphosphate) increased from 0.05 to 1.40 mM for the carboxylase reaction and by a similar amount for the oxygenase reaction. The Ki(O2) increased from 0.17 to 6.00 mM, but the ratio of carboxylase activity to oxygenase activity was scarcely affected by the change in amino acid. The binding of the transition state analogue 2-carboxyribitol 1,5-bisphosphate was reversible in the mutant and essentially irreversible in the wild type enzyme. Inhibition by fructose bisphosphate, competitive with ribulose bisphosphate, was slightly increased in the mutant enzyme. These data suggest that the change of the residue from methionine to leucine decreases the stability of the enediol reaction intermediate.
D001483:Base Sequence; D007700:Kinetics; D007930:Leucine; D008715:Methionine; D009154:Mutation; D010105:Oxygenases / Q000378:metabolism; D010428:Pentosephosphates / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D010428:Pentosephosphates; C049914:2-carboxyribitol 1,5-bisphosphate; D008715:Methionine; D010105:Oxygenases; D012273:Ribulose-Bisphosphate Carboxylase; D007930:Leucine