2-carboxy-d-hexitol 1,6-bisphosphate: An inhibitor of d-ribulose 1,5-bisphosphate carboxylase/oxygenase

2-Carboxy-d-hexitol 1,6-bisphosphate (CHBP) has been prepared from d-fructose 1,6-bisphosphate and cyanide. DEAE-Sephadex chromatography separated the reaction products into two fractions which were identified as CHBP and CHBP-lactone. CHBP is presumably a mixture of two diastereomers, 2-carboxy-d-glucitol 1,6-bisphosphate and 2-carboxy-d-mannitol 1,6-bisphosphate, but an attempt to separate these compounds was not successful. The material in the CHBP-lactone peak had no effect on d-ribulose 1,5-bisphosphate (RuBP) carboxylase. However, CHBP was a potent reversible inhibitor of RuBP carboxylases. This compound displayed an inhibition constant (K_i at pH 8.0 and 30 °C) of 1–2 μm with the enzymes from spinach and barley, while the K_i was 60–70 μm with bacterial RuBP carboxylases from Pseudomonas oxalaticus and Rhodospirillum rubrum. The mode of inhibition was competitive with respect to RuBP for all the carboxylases, and noncompetitive with respect to CO₂ for the enzymes from spinach, P. oxalaticus and R. rubrum. The results indicate that, in the binding of certain organic phosphates by RuBP carboxylases, there may be a fundamental difference between the enzymes isolated from microbial and from higher plant sources. RuBP oxygenase activities from spinach and P. oxalaticus were also inhibited by CHBP, with K_i values which were similar to those obtained with the carboxylase activity of the same enzymes. The mode of inhibition of the oxygenase activities was also competitive with respect to RuBP. Thus, it seems that the binding of CHBP is similar for the carboxylase and oxygenase reactions of the same enzyme.