J. Biol. Chem., Vol. 255, Issue 2, 509-511, Jan, 1980

Mass spectrometric analysis of the reactions of ribulosebisphosphate carboxylase/oxygenase

J Pierce, NE Tolbert and R Barker

The products of the reaction of D-[2-13C]ribulose 1,5-bisphosphate with molecular oxygen in the presence of D-ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed, after dephosphorylation, as the trimethylsilyl derivatives of glycolate and glycerate by mass spectrometry. The extent of isotopic incorporation into [1- 13C]glycolate from [1-13C]glycolate 2-phosphate produced in the oxidation reaction demonstrates that at least 95% of the glycolate 2- phosphate produced arises from carbon atoms 1 and 2 of D-ribulose 1,5- bisphosphate. When D-[2-18O]ribulose 1,5-bisphosphate was used, a significant amount of [1-18O]glycolate 2-phosphate was formed, indicating that O-2 of D-ribulose 1,5-bisphosphate is retained in the carboxyl oxygens of glycolate 2-phosphate. In addition, analyses of the products of the reaction between D-[2-13C]ribulose 1,5-bisphosphate and [13C]O2 confirm and extend the conclusions of an earlier report (Mullhofer, G., and Rose, I. A. (1965) J. Biol. Chem. 240, 1341-1346) that cleavage of D-ribulose 1,5-bisphosphate occurs between carbon atoms 2 and 3 during its enzymatic carboxylation. The results eliminate possible mechanisms involving the obligatory loss of O-2 of D-ribulose 1,5-bisphosphate during its enzymatic oxidation and confirm the specificity of carbon--carbon bond cleavage in both the oxygenase and carboxylase reactions.
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