Site-directed mutagenesis of lysine-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum
A variety of data suggest that Lys-329 of the title enzyme serves a catalytic function. To test this postulate, Lys-329 has been replaced with Gly, Ala, Ser, Cys, Arg, or Glu by a technique of site-directed mutagenesis that utilizes a suitably gapped plasmid carrying the target gene. The mutant proteins were produced in E. coli JM107 and purified to near homogeneity by immunoaffinity chromatography; they were shown to be dimers, like the wild-type enzyme, by gel electrophoresis. Hence, these amino acid substitutions are compatible with proper folding and association of subunits. The purified mutant proteins do not exhibit detectable enzyme activity nor do they form a stable quaternary complex with Mg/sup 2 +/, CO/sub 2/, and a transition-state analog as observed for wild-type enzyme. However, based on ligand-selective elution of the mutant proteins from an affinity matrix (green A agarose) they do retain the ability to bind substrate analogs. These results support an absolute essentiality of Lys-329; its precise function may be illuminated by further characterization of the mutant proteins.
- Research Organization:
- Oak Ridge National Lab., TN
- OSTI ID:
- 6279909
- Report Number(s):
- CONF-870644-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 46:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
Similar Records
Examination of the function of active site lysine 329 of ribulose-bisphosphate carboxylase/oxygenase as revealed by the proton exchange reaction
Reexamination of the binding site for pyridoxal 5'-phosphate in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLASE
CARBOXYLIC ACIDS
CELL CONSTITUENTS
ELECTROPHORESIS
ENZYMES
FUNCTIONS
GENES
KINETICS
LYASES
LYSINE
MICROORGANISMS
MUTANTS
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
OXYGENASES
PLASMIDS
REACTION KINETICS
RHODOSPIRILLUM