Intersubunit interaction at the active site of D-ribulose-1,5-bisphosphate carboxylase/oxygenase as revealed by cross-linking and site-directed mutagenesis
For measurement of distances between active-site residues of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum (a homodimer of 50.5-kDa subunits), the reaction of the enzyme with 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (which spans 9 A) has been explored. Inactivation of the enzyme by the bifunctional reagent is not associated with an increase in apparent molecular weight, thereby excluding intermolecular cross-linking. However, in the presence of urea, gel filtration of the inactivated enzyme reveals a prominent dimeric species attributed to intersubunit cross-linking. The major chromophoric peptide has been isolated from a tryptic digest of the dimer; sequence analysis of this peptide reveals that the intersubunit cross-link occurs between Cys-58 and active site Lys-166. In contrast to previous substitutions for Lys-166 introduced by site-directed mutagenesis, replacement by aspartic acid prevents association of the two subunits. In addition to identifying an intersubunit contact, these observations suggest that the catalytic site of the carboxylase is positioned at an interface between subunits and that segments of both subunits may be required for catalytic competence.
- Research Organization:
- Oak Ridge National Lab., TN
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 5968811
- Journal Information:
- Biochemistry; (United States), Vol. 26:15
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
CARBOXY-LYASES
AUTORADIOGRAPHY
CROSS-LINKING
MOLECULAR STRUCTURE
SULFONES
BIOLOGICAL EFFECTS
AMINO ACID SEQUENCE
ELECTROPHORESIS
INACTIVATION
LIQUID COLUMN CHROMATOGRAPHY
MUTAGENESIS
RHODOSPIRILLUM
BACTERIA
CARBON-CARBON LYASES
CHEMICAL REACTIONS
CHROMATOGRAPHY
ENZYMES
LYASES
MICROORGANISMS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
POLYMERIZATION
SEPARATION PROCESSES
560300* - Chemicals Metabolism & Toxicology
550200 - Biochemistry