Rubisco Mechanism: Dissection of the Enolization Partial Reaction. Final Report

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To test experimentally, the prior theoretical deduction that active-site residue Lys166 of ribulose-bisphosphate carboxylase participates in the carboxylation step of overall catalysis, site-directed mutants and chemically rescued site-directed mutants were characterized by kinetics and product analysis. Although position-166 mutants are able to catalyze normal enolization of ribulose bisphosphate, the enediol intermediate does not undergo carboxylation but rather eliminates phosphate. Furthermore, the chemically rescued mutant (aminoethylation of the severely impaired Lys66Cys mutant) generates a highly active mimic, which displays an enhanced carboxylation/oxygenation partition ratio. These two distinct lines of experimentation document a crucial role of Lys166 in carboxylation and in discrimination ... continued below

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Hartman, F. C. June 11, 2003.

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To test experimentally, the prior theoretical deduction that active-site residue Lys166 of ribulose-bisphosphate carboxylase participates in the carboxylation step of overall catalysis, site-directed mutants and chemically rescued site-directed mutants were characterized by kinetics and product analysis. Although position-166 mutants are able to catalyze normal enolization of ribulose bisphosphate, the enediol intermediate does not undergo carboxylation but rather eliminates phosphate. Furthermore, the chemically rescued mutant (aminoethylation of the severely impaired Lys66Cys mutant) generates a highly active mimic, which displays an enhanced carboxylation/oxygenation partition ratio. These two distinct lines of experimentation document a crucial role of Lys166 in carboxylation and in discrimination between CO{sub 2} and O{sub 2}. To ascertain whether Lys166 functions as an acid or base in facilitation of enolization, the chemically rescued mutant bearing {sup 15}N was titrated by NM R. From pH 6.5-9.5, the amino group of Lys166 remains unprotonated, indicating that it promotes enolization by hydrogen bonding to the ketone group of the substrate.

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OSTI as DE00824531

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  • Other Information: PBD: 11 Jun 2003

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  • Report No.: DOE/ER/20348-1
  • Grant Number: FG02-99ER20348
  • DOI: 10.2172/824531 | External Link
  • Office of Scientific & Technical Information Report Number: 824531
  • Archival Resource Key: ark:/67531/metadc780738

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  • June 11, 2003

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  • Dec. 3, 2015, 9:30 a.m.

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  • Aug. 4, 2016, 7:53 p.m.

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Hartman, F. C. Rubisco Mechanism: Dissection of the Enolization Partial Reaction. Final Report, report, June 11, 2003; United States. (digital.library.unt.edu/ark:/67531/metadc780738/: accessed August 17, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.