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

Conference · Fri May 01 00:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6375711

Lee, E H; Soper, T S; Hartman, F C

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.

• Research Organization: Oak Ridge National Lab., TN
• OSTI ID: 6375711
• Report Number(s): CONF-870644-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 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

Related Subjects

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