| Abstrakt: |
Site-directed mutagenesis has implicated active-site Lys329 of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in promoting the reaction of CO2 with the 2,3-enediol of ribulose bisphosphate and in stabilizing carboxylation intermediates [Hartman, F. C., & Lee, E. H. (1989) J. Biol. Chem. 264, 11784-11789; Lorimer, G. H., Chen, Y.-R., & Hartman, F. C. (1993) Biochemistry 32, 9018-9024]. Although the K329A mutant is greatly impaired in carboxylation, it catalyzes formation of the enediol, which is misprocessed to an O2-dependent side product [Harpel, M. R., & Hartman, F. C. (1994) Biochemistry 33, 5553-5561]. We now identify this novel side product as 2-carboxytetritol 1,4-bisphosphate (CTBP) by mass spectrometry, 1H-, 13C-, and 31P-NMR spectroscopy, and periodate oxidation. H2O2 accumulates during formation of CTBP, which we show to be derived from a transient precursor, the dicarbonyl D-glycero-2,3-pentodiulose 1,5-bisphosphate. The isolated dicarbonyl bisphosphate is processed by K329A to CTBP. These results, combined with isotope-labeling studies, suggest that CTBP arises by H2O2 elimination from an improperly stabilized peroxy adduct of the enediol intermediate, followed by rearrangement of the resulting dicarbonyl. Therefore, normal oxygenation, as catalyzed by wild-type Rubisco, is not a spontaneous reaction but must involve stabilization of the peroxy intermediate to mitigate formation of the dicarbonyl bisphosphate and subsequently CTBP. CTBP formation verifies the identity of Rubisco's previously invoked oxygenase intermediate, provides additional mechanistic insight into the oxygenation reaction, and shows that Lys329 promotes oxygenation as well as carboxylation. These results may be relevant to other oxygenases, which also exploit substrate carbanions rather than organic cofactors or transition metals for biological oxygen utilization. |
