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Title: Proton transfer reactions of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase

Abstract

The {epsilon}-amine of Lys166 of RuBP carboxylase/oxygenase facilitates enolization of RuBP (the initial step in overall catalysis) more than 100-fold, despite being too remote from C-3 of RuBP to be the primary proton acceptor. To clarify the roles of Lys166, we have analyzed products formed by mutants and chemically-rescued variants from both RuBP and isolated carboxylated reaction intermediate (CKABP). LysI66 mutants slowly convert RuBP to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, indicative of enolization followed by {beta}-elimination. Thus, Lys166 not only promotes enolization but forward processing of enediol as well. Restoration of carboxylase activity to K166C by aminoethylation increases CO{sub 2}/O{sub 2} specificity and also enhances pyruvate formation, which reflects {beta}-elimination of the terminal aci-acid intermediate. The previously uncharacterized product of CKABP hydrolysis by K166G is shown to be pyruvate, rather than 3-phosphoglycerate, as normally formed by protonation of the terminal intermediate. These results are consistent with Lys166 as the terminal proton donor, as deduced by crystallography, and demonstrate multiple roles for this residue in catalysis.

Authors:
;  [1]
  1. Oak Ridge National Lab., TN (United States)
Publication Date:
OSTI Identifier:
538944
Report Number(s):
CONF-960807-
TRN: 97:004029-0011
Resource Type:
Conference
Resource Relation:
Conference: 212. national meeting of the American Chemical Society (ACS), Orlando, FL (United States), 25-30 Aug 1996; Other Information: PBD: 1996; Related Information: Is Part Of 212th ACS national meeting; PB: 1830 p.
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; CARBOXYLASE; ENZYME ACTIVITY; OXYGENASES; CATALYSIS; CRYSTALLOGRAPHY; HYDROLYSIS; MUTANTS; RIBULOSE; REACTION INTERMEDIATES; LYSINE

Citation Formats

Harpel, M R, and Hartman, F C. Proton transfer reactions of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. United States: N. p., 1996. Web.
Harpel, M R, & Hartman, F C. Proton transfer reactions of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase. United States.
Harpel, M R, and Hartman, F C. Tue . "Proton transfer reactions of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase". United States.
@article{osti_538944,
title = {Proton transfer reactions of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase},
author = {Harpel, M R and Hartman, F C},
abstractNote = {The {epsilon}-amine of Lys166 of RuBP carboxylase/oxygenase facilitates enolization of RuBP (the initial step in overall catalysis) more than 100-fold, despite being too remote from C-3 of RuBP to be the primary proton acceptor. To clarify the roles of Lys166, we have analyzed products formed by mutants and chemically-rescued variants from both RuBP and isolated carboxylated reaction intermediate (CKABP). LysI66 mutants slowly convert RuBP to 1-deoxy-D-glycero-2,3-pentodiulose 5-phosphate, indicative of enolization followed by {beta}-elimination. Thus, Lys166 not only promotes enolization but forward processing of enediol as well. Restoration of carboxylase activity to K166C by aminoethylation increases CO{sub 2}/O{sub 2} specificity and also enhances pyruvate formation, which reflects {beta}-elimination of the terminal aci-acid intermediate. The previously uncharacterized product of CKABP hydrolysis by K166G is shown to be pyruvate, rather than 3-phosphoglycerate, as normally formed by protonation of the terminal intermediate. These results are consistent with Lys166 as the terminal proton donor, as deduced by crystallography, and demonstrate multiple roles for this residue in catalysis.},
doi = {},
url = {https://www.osti.gov/biblio/538944}, journal = {},
number = ,
volume = ,
place = {United States},
year = {1996},
month = {12}
}

Conference:
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