Interaction of ribulosebisphosphate carboxylase/oxygenase with transition-state analogues

2-C-Carboxy-D-ribitol 1,5-bisphosphate and 2-C-carboxy-D-arabinitol 1,5-bisphosphate have been synthesized, purified, and characterized. In the presence of Mg2+, 2-C-carboxy-D-arabinitol 1,5-bisphosphate binds to ribulose-1,5-bisphosphate carboxylase/oxygenase by a two-step mechanism. The first, rapid step is similar to the binding of ribulose 1,5-bisphosphate or its structural analogues. The second step is a slower process (k = 0.04 s-1) and accounts for the tighter binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (Kd less than or approximately to 10(-11) M) than of 2-C-carboxy-D-ribitol 1,5-bisphosphate (Kd = 1.5 X 10(6) M). Both carboxypentitol bisphosphates exhibit competitive inhibition with respect to ribulose 1,5-bisphosphate. 2-C-(Hydroxymethyl)-D-ribitol 1,5-bisphosphate and 2-C-(hydroxymethyl)-D-arabinitol 1,5-bisphosphate were also synthesized; both are competitive inhibitors with respect to ribulose 1,5-bisphosphate with Ki = 8.0 X 10(-5) M and Ki = 5.0 X 10(-6) M, respectively. Thus, the carboxyl group of 2-C-carboxy-D-arabinitol 1,5-bisphosphate is necessary for maximal interaction with the enzyme. Additionally, Mg2+ is essential for the tight binding of 2-C-carboxy-D-arabinitol 1,5-bisophsphate. A model for catalysis of ribulose 1,5-bisphosphate carboxylation is discussed which includes a functional role for Mg2+ in the stabilization of the intermediate 2-C-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. Mechanistic implications that arise from the stereochemistry of this intermediate are also discussed.