Beta-elimination of phosphate from reaction intermediates by site-directed mutants of ribulose-bisphosphate carboxylase/oxygenase.
Five residues (Thr-53, Asn-54, Gly-370, Gly-393, and Gly-394) of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase are positioned to serve as hydrogen-bond donors for the C1 phosphate of ribulose bisphosphate and thereby constrain conformational flexibility of the initial enediol(ate) intermediate (Knight, S., Andersson, I., and Brändén, C.-I. (1990) J. Mol. Biol. 215, 113-160). To study the functional contributions of the residues implicated in ribulose bisphosphate binding and intermediate stabilization, we have replaced them individually with alanine, either to remove the H-bonding group (T53A, N54A) or to introduce bulk (G370A, G393A, G394A). Consequences of substitutions include diminution of carboxylase activity (with a lesser impact on enolization activity), increase of Km (ribulose bisphosphate), and decrease of carboxylation: oxygenation specificity. During catalytic turnover of ribulose bisphosphate by several mutants, substantial amounts of the substrate are diverted to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, reflecting beta-elimination of phosphate from the enediol(ate) intermediate. This side product is not observed with wild-type enzyme, nor has it been reported with mutant enzymes characterized previously. Another consequence of disruption of the phosphate binding site is enhanced production of pyruvate, relative to wild-type enzyme, by some of the mutants due to decomposition of the acicarbanion of 3-phosphoglycerate (the terminal intermediate). These data provide direct evidence that phosphate ligands stabilize conformations of intermediates that favor productive turnover and mitigate beta-elimination at two stages of overall catalysis.
Larimer
F W
FW
Protein Engineering Program, Oak Ridge National Laboratory, Tennessee 37831-8077.
Harpel
M R
MR
Hartman
F C
FC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010710:Phosphates / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012274:Ribulosephosphates / Q000737:chemistry / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D011994:Recombinant Proteins; D012274:Ribulosephosphates; C001933:ribulose-1,5 diphosphate; D012273:Ribulose-Bisphosphate Carboxylase