Multiple catalytic roles of His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase/oxygenase.
Active-site His 287 of Rhodospirillum rubrum ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase interacts with the C3-hydroxyl of bound substrate or reaction-intermediate analogue (CABP), water molecules, and ligands for the activator metal-ion (Andersson I, 1996, J Mol Biol 259:160-174; Taylor TC, Andersson I, 1997, J Mol Biol 265:432-444). To test structure-based postulates of catalytic functionality, His 287 was replaced with Asn or Gln. The mutants are not affected adversely in subunit assembly, activation (binding of Mg2+ and carbamylation of Lys 191), or recognition of phosphorylated ligands; they bind CABP with even greater tenacity than does wild-type enzyme. H287N and H287Q are severely impaired in catalyzing overall carboxylation (approximately 10(3)-fold and > 10(5)-fold, respectively) and enolization (each mutant below threshold for detection) of RuBP. H287N preferentially catalyzes decarboxylation of carboxylated reaction intermediate instead of forward processing to phosphoglycerate. Analysis of RuBP turnover that occurs at high concentrations of mutants over extended time periods reveal > 10-fold reduced CO2/O2 specificities, elevated misprotonation of the enediol intermediate, and misprocessing of the oxygenated intermediate of the oxygenase pathway. These results are consistent with multifaceted roles for His 287 in promoting enediol formation, enediol tautomerization, and forward-processing of carboxylated intermediate.
Harpel
M R
MR
Protein Engineering Program, Life Sciences Division, Oak Ridge National Laboratory, Tennessee 37831-8080, USA.
Larimer
F W
FW
Hartman
F C
FC
D002384:Catalysis; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry*; D013329:Structure-Activity Relationship
D008667:Metalloproteins; D006639:Histidine; D012273:Ribulose-Bisphosphate Carboxylase