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."[1]References
- The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase. Lorimer, G.H., Gutteridge, S., Reddy, G.S. J. Biol. Chem. (1989) [Pubmed]
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