The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase.
There are four possible orientations of the substrate ribulose 1,5-bisphosphate in the active site of ribulose-1,5-bisphosphate carboxylase. Distinction between these four possible orientations has been made on the basis of 31P NMR and borohydride-trapping experiments. The orientation of the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate with respect to the divalent metal ion was determined by 31P NMR studies of the quaternary complex, enzyme.CO2.Ni2+.2'-carboxyarabinitol 1,5-bisphosphate. Assignment of the phosphorus resonances of this complex was made by labeling the phosphoryl group at either C-1 or C-5 with 17O. The phosphorus atom closer to the paramagnetic metal ion, Ni2+, to which the broader of the phosphorus resonances is attributed, has been identified as that attached to C-1. When bound to the active site of carbaminated enzyme, D-ribulose 1,5-bisphosphate was reduced by sodium borohydride with absolute stereospecificity to D-arabinitol 1,5-bisphosphate. The reduction of the enzyme-bound substrate thus occurred on the Si face of the C-2 carbonyl group. These two results together establish that ribulose 1,5-bisphosphate is oriented within the active site so that 1) the phosphoryl group at C-1 is closer to the divalent metal ion than that at C-5 and 2) the Si face of the carbonyl group points to the "outside world."
Lorimer
G H
GH
Central Research and Developmental Department, E. I. du Pont de Nemours and Company, Wilmington, Delaware 19880.
Gutteridge
S
S
Reddy
G S
GS
D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008968:Molecular Conformation; D010758:Phosphorus; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D010758:Phosphorus; D012273:Ribulose-Bisphosphate Carboxylase