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Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

Thesis/Dissertation ·
OSTI ID:7140832
The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with (/sup 14/C)pyruvate in the presence of NaCNBH/sub 3/ results in the incorporation of one mol of /sup 14/C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with (3-/sup 14/C)bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved.
Research Organization:
Michigan Univ., Ann Arbor (USA)
OSTI ID:
7140832
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550701 -- Microbiology-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDE-LYASES
ALDOLASE
ALKYLATION
AMINO ACID SEQUENCE
AMINO ACIDS
ARGININE
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CARBON-CARBON LYASES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CYSTEINE
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
ESCHERICHIA COLI
ESTERIFICATION
HYDROLASES
ISOTOPE APPLICATIONS
KETO ACIDS
KINETICS
LABELLED COMPOUNDS
LYASES
LYSINE
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC BROMINE COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC HALOGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDE HYDROLASES
PYRUVIC ACID
REACTION KINETICS
SERINE PROTEINASES
THIOLS
TRACER TECHNIQUES
TRYPSIN