Facilitation of the Terminal Proton Transfer Reaction of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase by Active-Site Lys166
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- 作者：Mark R. Harpel and Fred C. Hartman
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：November 5, 1996
- 年：1996
- 卷：35
- 期：44
- 页码：13865 - 13870
- 全文大小：312K
- 年卷期：v.35,no.44(November 5, 1996)
- ISSN：1520-4995
文摘
The terminal step in the carboxylation pathway catalyzed byribulose 1,5-bisphosphatecarboxylase/oxygenase (Rubisco) is stereospecific protonation of theC-2 aci-acid of 3-phosphoglycerate(PGA). X-ray crystallographic results favor the 
-amino group ofLys166 as the proton donor in this step[Knight et al. (1990) J. Mol. Biol. 215, 113].Nonetheless, position-166 mutants are able tocatalyzeforward processing of isolated 2-carboxy-3-ketoarabinitol1,5-bisphosphate (CKABP), the carboxylatedreaction intermediate [Lorimer, G. H., & Hartman, F. C. (1988)J. Biol. Chem. 263, 6468]. Priorassaysfor intermediate processing relied solely on formation of acid-stableradioactivity from acid-labile [2'-14C]CKABP. Therefore, PGA, the normal reactionproduct, may not have been distinguished from pyruvate,the product from 
-elimination of phosphate from the terminalaci-acid intermediate [Andrews, T. J., &Kane, H. J. (1991) J. Biol. Chem. 266, 9447].If Lys166 indeed serves as the terminal proton donor,mutants lacking an ionizable side chain at position 166 might processthe carboxylated intermediatepredominantly to pyruvate. We have thus used anion exchangechromatography and enzyme coupling toseparate and identify the products from turnover of[2'-14C]CKABP by wild-type, K166G, andK166Senzymes. Although PGA is the only labeled product of significanceformed by wild-type enzyme, pyruvateis a major labeled product formed by the mutants. These resultsprovide the first direct functionally-based evidence that Lys166 is crucial to the last step inRubisco-catalyzed conversion of RuBP to PGA.
-amino group ofLys166 as the proton donor in this step[Knight et al. (1990) J. Mol. Biol. 215, 113].Nonetheless, position-166 mutants are able tocatalyzeforward processing of isolated 2-carboxy-3-ketoarabinitol1,5-bisphosphate (CKABP), the carboxylatedreaction intermediate [Lorimer, G. H., & Hartman, F. C. (1988)J. Biol. Chem. 263, 6468]. Priorassaysfor intermediate processing relied solely on formation of acid-stableradioactivity from acid-labile [2'-14C]CKABP. Therefore, PGA, the normal reactionproduct, may not have been distinguished from pyruvate,the product from -elimination of phosphate from the terminalaci-acid intermediate [Andrews, T. J., &Kane, H. J. (1991) J. Biol. Chem. 266, 9447].If Lys166 indeed serves as the terminal proton donor,mutants lacking an ionizable side chain at position 166 might processthe carboxylated intermediatepredominantly to pyruvate. We have thus used anion exchangechromatography and enzyme coupling toseparate and identify the products from turnover of[2'-14C]CKABP by wild-type, K166G, andK166Senzymes. Although PGA is the only labeled product of significanceformed by wild-type enzyme, pyruvateis a major labeled product formed by the mutants. These resultsprovide the first direct functionally-based evidence that Lys166 is crucial to the last step inRubisco-catalyzed conversion of RuBP to PGA.