Site-specific mutagenesis of ribulose-1,5-bisphosphate carboxylase/oxygenase. Evidence that carbamate formation at Lys 191 is required for catalytic activity.
Site-specific mutagenesis of a cloned gene for ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was used to examine the functional significance of carbamate activation. Lysine 191, the residue involved in carbamate formation, was replaced with a glutamate in order to mimic the anionic nature of the carbamate. The resulting enzyme was capable of binding the six-carbon transition state analog carboxyarabinitol bisphosphate, but completely lacked catalytic activity. In contrast to the wild-type enzyme, carboxyarabinitol bisphosphate binding was not stabilized by divalent metal and CO2. These observations are consistent with a proposed role for the carbamate in binding the metal required for catalysis.
Estelle
M
M
Hanks
J
J
McIntosh
L
L
Somerville
C
C
D000595:Amino Acid Sequence; D002219:Carbamates / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D008239:Lysine / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D009154:Mutation; D010428:Pentosephosphates; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*; D013329:Structure-Activity Relationship; D013402:Sugar Alcohols; D013403:Sugar Phosphates / Q000378:metabolism
D002219:Carbamates; D010428:Pentosephosphates; D013402:Sugar Alcohols; D013403:Sugar Phosphates; D002245:Carbon Dioxide; C032687:2-carboxyarabinitol 1,5-biphosphate; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium; D008239:Lysine