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Title: Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O{sub 2}-dependent side product generated by a site-directed mutant

Abstract

Site-directed mutagenesis has implicated active-site Lys329 of Rhodospirillum rubrum ribulose-1.5-bisphosphate carboxylase/oxygenase (Rubisco) in promoting the reaction of CO{sub 2} with the 2,3-enediol of ribulose bisphosphate and in stabilizing carboxylation intermediates. Although the K329A mutant is greatly impaired in carboxylation, it catalyzes formation of the enediol, which is misprocessed to an O{sub 2}-dependent side product. We now identify this novel side product as 2-carboxytetritol 1,4-bisphosphate (CTBP) by mass spectrometry, {sup 1}H-, {sup 13}C-, and {sup 31}P-NMR spectroscopy, and periodate oxidation. H{sub 2}O{sub 2} accumulates during formation of CTBP, which we show to be derived form 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 H{sub 2}O{sub 2} 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 asmore » 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. 63 refs., 10 figs., 2 tabs.« less

Authors:
;  [1];  [2]
  1. Oak Ridge National Laboratory, TN (United States)
  2. Univ. of Tennessee, Knoxville, TN (United States); and others
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
191881
Resource Type:
Journal Article
Journal Name:
Biochemistry (Eaton)
Additional Journal Information:
Journal Volume: 34; Journal Issue: 35; Other Information: PBD: 5 Sep 1995
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; OXYGENASES; ENZYME ACTIVITY; MUTAGENESIS; OXIDATION; CARBOXYLATION; PRECURSOR; RHODOSPIRILLUM; RIBULOSE; SPECTROSCOPY; REACTION INTERMEDIATES

Citation Formats

Harpel, M R, Hartman, F C, and Serpersu, E H. Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O{sub 2}-dependent side product generated by a site-directed mutant. United States: N. p., 1995. Web. doi:10.1021/bi00035a039.
Harpel, M R, Hartman, F C, & Serpersu, E H. Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O{sub 2}-dependent side product generated by a site-directed mutant. United States. https://doi.org/10.1021/bi00035a039
Harpel, M R, Hartman, F C, and Serpersu, E H. Tue . "Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O{sub 2}-dependent side product generated by a site-directed mutant". United States. https://doi.org/10.1021/bi00035a039.
@article{osti_191881,
title = {Oxygenation mechanism of ribulose-bisphosphate carboxylase/oxygenase. Structure and origin of 2-carboxytetritol 1,4-bisphosphate, a novel O{sub 2}-dependent side product generated by a site-directed mutant},
author = {Harpel, M R and Hartman, F C and Serpersu, E H},
abstractNote = {Site-directed mutagenesis has implicated active-site Lys329 of Rhodospirillum rubrum ribulose-1.5-bisphosphate carboxylase/oxygenase (Rubisco) in promoting the reaction of CO{sub 2} with the 2,3-enediol of ribulose bisphosphate and in stabilizing carboxylation intermediates. Although the K329A mutant is greatly impaired in carboxylation, it catalyzes formation of the enediol, which is misprocessed to an O{sub 2}-dependent side product. We now identify this novel side product as 2-carboxytetritol 1,4-bisphosphate (CTBP) by mass spectrometry, {sup 1}H-, {sup 13}C-, and {sup 31}P-NMR spectroscopy, and periodate oxidation. H{sub 2}O{sub 2} accumulates during formation of CTBP, which we show to be derived form 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 H{sub 2}O{sub 2} 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. 63 refs., 10 figs., 2 tabs.},
doi = {10.1021/bi00035a039},
url = {https://www.osti.gov/biblio/191881}, journal = {Biochemistry (Eaton)},
number = 35,
volume = 34,
place = {United States},
year = {1995},
month = {9}
}