A SIGNATURE OF THE OXYGENASE INTERMEDIATE IN CATALYSIS BY RIBULOSE-BISPHOSPHATE CARBOXYLASE OXYGENASE AS PROVIDED BY A SITE-DIRECTED MUTANT

被引:21
作者
CHEN, YR
HARTMANN, FC
机构
[1] OAK RIDGE NATL LAB,DIV BIOL,PROT ENGN PROGRAM,OAK RIDGE,TN 37831
[2] UNIV TENNESSEE,GRAD SCH BIOMED SCI,OAK RIDGE,TN 37831
关键词
D O I
10.1074/jbc.270.20.11741
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
An uncharacterized minor transient product, observed in our earlier studies of substrate turnover by the E48Q mutant of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase (Lee, E. H., Harpel, M. R., Chen, Y. R., and Hartman, F. C. (1993) J. Biol. Chem. 268, 26583-26591), becomes a major product when it is trapped and stabilized with berate as an additive to the reaction mixture. Chemical characterization establishes this novel product as D-glycero-2,3-pentodiulose 1,5-bisphosphate, thereby demonstrating oxidation of the C-3 hydroxyl of D- ribulose 1,5-bisphosphate to a carbonyl. As the formation of the novel oxidation product is oxygen-dependent and generates hydrogen peroxide, its precursor must be a peroxy derivative of ribulose bisphosphate. Thus, discovery of the dicarbonyl bisphosphate lends direct support to the long standing, but heretofore unproven, postulate that the normal pathway for oxidative cleavage of ribulose bisphosphate by the wild-type enzyme entails a peroxy intermediate. Our results also suggest that stabilization of the peroxy intermediate by the wild-type enzyme promotes carbon-carbon scission as opposed to elimination of hydrogen peroxide.
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页码:11741 / 11744
页数:4
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