Chen Y R, Hartman F C
University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences 37831, USA.
J Biol Chem. 1995 May 19;270(20):11741-4. doi: 10.1074/jbc.270.20.11741.
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 borate 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.
在我们早期对红螺菌核酮糖-1,5-二磷酸羧化酶/加氧酶E48Q突变体的底物周转研究中观察到一种未鉴定的次要瞬时产物（Lee, E. H., Harpel, M. R., Chen, Y.-R., and Hartman, F. C. (1993) J. Biol. Chem. 268, 26583 - 26591），当在反应混合物中添加硼酸盐将其捕获并稳定时，该产物成为主要产物。化学表征确定这种新产物为D-甘油-2,3-戊二酮糖1,5-二磷酸，从而证明了D-核酮糖-1,5-二磷酸的C-3羟基被氧化为羰基。由于这种新氧化产物的形成依赖于氧气并产生过氧化氢，其前体必定是核酮糖二磷酸的过氧衍生物。因此，二羰基二磷酸的发现直接支持了长期存在但迄今未得到证实的假设，即野生型酶氧化裂解核酮糖二磷酸的正常途径需要一个过氧中间体。我们的结果还表明，野生型酶对过氧中间体的稳定作用促进了碳-碳断裂，而不是过氧化氢的消除。
