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Title: Subunit interactions of ribulose bisphosphate carboxylase/oxygenase as determined by chemical cross-linking and site-directed mutagenesis

Abstract

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 species of 100-kDa, attributed to intersubunit cross-linking. The major chromophoric peptide has been isolated from a tryptic digest of the 100-kDa material. Sequence analyses reveal that the intersubunit cross-link occurs between Cys-58 and active-site Lys-166. In contrast to other substitutions by site-directed mutagenesis, replacement of Lys-166 with Asp prevents association of the two subunits, presumably as a consequence of repulsive forces between the thiolate and carboxylate anions. These observations suggest that each catalytic site may consist of residues from both subunits.

Authors:
; ;
Publication Date:
Research Org.:
Oak Ridge National Lab., TN
OSTI Identifier:
6375711
Report Number(s):
CONF-870644-
Journal ID: CODEN: FEPRA
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CARBOXYLASE; MOLECULAR STRUCTURE; OXYGENASES; AMINO ACID SEQUENCE; CROSS-LINKING; MOLECULAR WEIGHT; RHODOSPIRILLUM; STILBENE; AROMATICS; BACTERIA; CARBON-CARBON LYASES; CARBOXY-LYASES; CHEMICAL REACTIONS; ENZYMES; HYDROCARBONS; LYASES; MICROORGANISMS; ORGANIC COMPOUNDS; OXIDOREDUCTASES; POLYMERIZATION; 550200* - Biochemistry

Citation Formats

Lee, E H, Soper, T S, and Hartman, F C. Subunit interactions of ribulose bisphosphate carboxylase/oxygenase as determined by chemical cross-linking and site-directed mutagenesis. United States: N. p., 1987. Web.
Lee, E H, Soper, T S, & Hartman, F C. Subunit interactions of ribulose bisphosphate carboxylase/oxygenase as determined by chemical cross-linking and site-directed mutagenesis. United States.
Lee, E H, Soper, T S, and Hartman, F C. Fri . "Subunit interactions of ribulose bisphosphate carboxylase/oxygenase as determined by chemical cross-linking and site-directed mutagenesis". United States.
@article{osti_6375711,
title = {Subunit interactions of ribulose bisphosphate carboxylase/oxygenase as determined by chemical cross-linking and site-directed mutagenesis},
author = {Lee, E H and Soper, T S and Hartman, F C},
abstractNote = {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 species of 100-kDa, attributed to intersubunit cross-linking. The major chromophoric peptide has been isolated from a tryptic digest of the 100-kDa material. Sequence analyses reveal that the intersubunit cross-link occurs between Cys-58 and active-site Lys-166. In contrast to other substitutions by site-directed mutagenesis, replacement of Lys-166 with Asp prevents association of the two subunits, presumably as a consequence of repulsive forces between the thiolate and carboxylate anions. These observations suggest that each catalytic site may consist of residues from both subunits.},
doi = {},
url = {https://www.osti.gov/biblio/6375711}, journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 46:6,
place = {United States},
year = {1987},
month = {5}
}

Conference:
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