Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding
Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.
- Research Organization:
- Rice Univ., Houston, TX (USA)
- OSTI ID:
- 6908823
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:7; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AZOLES
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
DNA
ESCHERICHIA COLI
FORMATES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
IMIDAZOLES
INHIBITION
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LYSINE
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOPROTEINS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PROTEINS
REACTION KINETICS
SPECTROPHOTOMETRY
TRACER TECHNIQUES
TRANSCRIPTION FACTORS