Mutations of an active site threonyl residue promote beta elimination and other side reactions of the enediol intermediate of the ribulosebisphosphate carboxylase reaction.
The side chain of residue threonine 65 within the active site of ribulosebisphosphate carboxylase participates in a network of hydrogen bonds and ionic interactions involving the phosphate moiety attached to C-1 of the substrate. This residue was replaced with serine, alanine, and valine in the enzyme from Synechococcus PCC 6301. The mutant enzymes were stable, expressed abundantly by Escherichia coli, and retained the ability to form gel-filterable complexes with the reaction-intermediate analog, 2'-carboxyarabinitol-1,5-bisphosphate. The substitutions reduced the kcat/Km(CO2) (where kcat is the substrate-saturated turnover rate) of the enzyme from 17- to 340-fold with the more radical substitutions causing more severe reductions. The CO2/O2 specificity also deteriorated progressively, the valine replacement causing a 2.3-fold reduction. In concert with these changes, a compound tentatively identified as 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate, the product of beta elimination of the 2,3-enediol(ate) intermediate of the catalytic reaction, appeared among the reaction products in progressively increasing amounts. In the case of the valine substitution, it comprised 13% of the ribulose bisphosphate consumed. The mutant enzymes also partitioned more of their reaction flux to pentulose bisphosphate isomers of ribulose bisphosphate. By contrast, the diversion of carboxylated product to pyruvate, as a result of beta elimination of the three-carbon aci-carbanion intermediate of the carboxylation reaction, was ameliorated by the replacements, the valine mutant showing a 5-fold improvement in this parameter. These observations focus attention on a geometric conflict which exists between the requirements for stabilization of the 5-carbon enediol(ate) and 3-carbon aci-carbanion intermediates. This conflict must be resolved by a change in the angle of the C-1/bridge oxygen bond during each catalytic cycle. The network of hydrogen bonds involving the side chain of threonine 65 must play a crucial role in facilitating reaction of the enediol(ate) with the gaseous substrate and in shepherding this subsequent movement.
Morell
M K
MK
Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, Canberra.
Paul
K
K
O'Shea
N J
NJ
Kane
H J
HJ
Andrews
T J
TJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D004926:Escherichia coli; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013912:Threonine
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013912:Threonine; D012273:Ribulose-Bisphosphate Carboxylase