OXYGENATION MECHANISM OF RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE - STRUCTURE AND ORIGIN OF 2-CARBOXYTETRITOL 1,4-BISPHOSPHATE, A NOVEL O-2-DEPENDENT SIDE PRODUCT GENERATED BY A SITE-DIRECTED MUTANT

Site-directed mutagenesis has implicated active-site Lys329 of Rhodosp irillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisc o) in promoting the reaction of CO2 with the 2,3-enediol of ribulose b isphosphate 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 ca talyzes formation of the enediol, which is misprocessed to an O-2-depe ndent side product [Harpel, M. R., & Hartman, F. C. (1994) Biochemistr y 33, 5553-5561]. We now identify this novel side product as 2-carboxy tetritol 1,4-bisphosphate (CTBP) by mass spectrometry, H-1-, C-13-, an d P-31-NMR spectroscopy, and periodate oxidation. H2O2 accumulates dur ing formation of CTBP, which we show to be derived from a transient pr ecursor, the dicarbonyl D-glycero-2,3-pentodiulose 1,5-bisphosphate. T he isolated dicarbonyl bisphosphate is processed by K329A to CTBP. The se 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 resulti ng dicarbonyl. Therefore, normal oxygenation, as catalyzed by wild-typ e Rubisco, is not a spontaneous reaction but must involve stabilizatio n of the peroxy intermediate to mitigate formation of the dicarbonyl b isphosphate and subsequently CTBP. CTBP formation verifies the identit y of Rubisco's previously invoked oxygenase intermediate, provides add itional mechanistic insight into the oxygenation reaction, and shows t hat Lys329 promotes oxygenation as well as carboxylation. These result s may be relevant to other oxygenases, which also exploit substrate ca rbanions rather than organic cofactors or transition metals for biolog ical oxygen utilization.