Function of active-site residues of ribulose bisphosphate carboxylase/oxygenase

Title: Function of active-site residues of ribulose bisphosphate carboxylase/oxygenase

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Abstract

As one facet of elucidating the mechanism of ribulose-P/sub 2/ carboxylase and of ultimately evaluating the feasibility of altering the carboxylase/oxygenase ratio, active-site characterization has received considerable attention. Chemical modification has revealed five disparate segments of primary structure that appear to constitute the active site. The amino acid sequence of each segment is highly conserved among evolutionarily diverse carboxylases. Site-directed mutagenesis has been used to clarify further the functions of Lys-166, Lys-329, His-291, and Glu-48 of ribulose-P/sub 2/ carboxylase from Rhodospirillum rubrum. These two lysines were selected for examination because of the wealth of data discussed earlier that had implicated their catalytic involvement. His-291 was scrutinized because of the suggestion of its participation, rather than Lys-166, as the proton transfer group that enolizes ribulose-P/sub 2/. The reasons for inspecting Glu-48 were circumstantial. Within the homologous region flanked by His-44 and Cys-58, targets of an affinity label, Glu-48 is the only acid/base group and hence a logical candidate for functionality. Furthermore, crystallographic and chemical cross-linking studies had placed this NH/sub 2/-terminal region near the active site. 33 refs.

Authors:: Hartman, F C; Foote, R S; Larimer, F W; Lee, E H; Machanoff, R; Milanez, S; Mitra, S; Mural, R J; Niyogi, S K; Smith, H B

Publication Date:: 1987-01-01

Research Org.:: Oak Ridge National Lab., TN (USA)

OSTI Identifier:: 6432826

Report Number(s):: CONF-8706127-1
ON: DE87011124

DOE Contract Number:: AC05-84OR21400

Resource Type:: Conference

Resource Relation:: Conference: Plant molecular biology, Copenhagen, Denmark, 10 Jun 1987

Country of Publication:: United States

Language:: English

Subject:: 14 SOLAR ENERGY; 59 BASIC BIOLOGICAL SCIENCES; RIBULOSE DIPHOSPHATE CARBOXYLASE; BIOCHEMICAL REACTION KINETICS; GENE MUTATIONS; STRUCTURE-ACTIVITY RELATIONSHIPS; AMINO ACID SEQUENCE; BIOMIMETIC PROCESSES; SPECIES DIVERSITY; SPINACH; CARBON-CARBON LYASES; CARBOXY-LYASES; ENZYMES; FOOD; KINETICS; LYASES; MOLECULAR STRUCTURE; MUTATIONS; PLANTS; REACTION KINETICS; VEGETABLES; 140505* - Solar Energy Conversion- Photochemical, Photobiological, & Thermochemical Conversion- (1980-); 550200 - Biochemistry

Citation Formats


                    Hartman, F C, Foote, R S, Larimer, F W, Lee, E H, Machanoff, R, Milanez, S, Mitra, S, Mural, R J, Niyogi, S K, and Smith, H B. Function of active-site residues of ribulose bisphosphate carboxylase/oxygenase.  United States: N. p., 1987. 
        Web.

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                    Hartman, F C, Foote, R S, Larimer, F W, Lee, E H, Machanoff, R, Milanez, S, Mitra, S, Mural, R J, Niyogi, S K, & Smith, H B. Function of active-site residues of ribulose bisphosphate carboxylase/oxygenase.  United States.

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                    Hartman, F C, Foote, R S, Larimer, F W, Lee, E H, Machanoff, R, Milanez, S, Mitra, S, Mural, R J, Niyogi, S K, and Smith, H B. Thu .  
        "Function of active-site residues of ribulose bisphosphate carboxylase/oxygenase".  United States.

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@article{osti_6432826,

  title        = {Function of active-site residues of ribulose bisphosphate carboxylase/oxygenase},

  author       = {Hartman, F C and Foote, R S and Larimer, F W and Lee, E H and Machanoff, R and Milanez, S and Mitra, S and Mural, R J and Niyogi, S K and Smith, H B},

  abstractNote = {As one facet of elucidating the mechanism of ribulose-P/sub 2/ carboxylase and of ultimately evaluating the feasibility of altering the carboxylase/oxygenase ratio, active-site characterization has received considerable attention. Chemical modification has revealed five disparate segments of primary structure that appear to constitute the active site. The amino acid sequence of each segment is highly conserved among evolutionarily diverse carboxylases. Site-directed mutagenesis has been used to clarify further the functions of Lys-166, Lys-329, His-291, and Glu-48 of ribulose-P/sub 2/ carboxylase from Rhodospirillum rubrum. These two lysines were selected for examination because of the wealth of data discussed earlier that had implicated their catalytic involvement. His-291 was scrutinized because of the suggestion of its participation, rather than Lys-166, as the proton transfer group that enolizes ribulose-P/sub 2/. The reasons for inspecting Glu-48 were circumstantial. Within the homologous region flanked by His-44 and Cys-58, targets of an affinity label, Glu-48 is the only acid/base group and hence a logical candidate for functionality. Furthermore, crystallographic and chemical cross-linking studies had placed this NH/sub 2/-terminal region near the active site. 33 refs.},

  doi          = {},

  url          = {https://www.osti.gov/biblio/6432826},
  journal      = {},
number       = ,

  volume       = ,

  place        = {United States},

  year         = {1987},

  month        = {1}

}

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Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction

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Diverse approaches that include site-directed mutagenesis have indicated a catalytic role of Lys-329 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. To determine whether Lys-329 is required for the initial enolization of ribulose bisphosphate or for some subsequent step in the overall reaction pathway, the competence of position 329 mutant proteins (devoid of carboxylase activity) in catalyzing exchange of solvent protons with the C-3 proton of substrate has now been examined. Irrespective of the amino acid substitution for Lys-329, the mutant protein retains 2-6% of the wild-type activity in the proton exchange reaction. The complete stability of ribulose bisphosphate during the enolizationmore »« less
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Ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is a homodimer of 50-kDa subunits. Although a high-resolution 3D structure of the enzyme has not yet been reported, recent work from their laboratory with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrated an intrasubunit distance of 12 A between the active-site lysines at positions 166 and 329. To continue mapping distances between residues in the active-site region, they have modified the enzyme with 4,4'-difluoro-3,3'-dinitrophenyl sulfone, which spans 9 A. The inactivated carboxylase exhibits an unaltered molecular weight as judged by gel filtration, thereby excluding intermolecular cross-linking. However, in the presence of urea, gel filtration reveals a prominent speciesmore »« less
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