Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase
- North Texas State Univ., Denton
N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo(/sup 14/C/sub 2/)acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.
- DOE Contract Number:
- W-7405-ENG-26
- OSTI ID:
- 6795635
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 255:19
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ISOMERASES
HISTIDINE
LABELLING
MOLECULAR STRUCTURE
ACID HYDROLYSIS
AFFINITY
AMINO ACIDS
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
ENZYME ACTIVITY
EXPERIMENTAL DATA
GLUCOSE
ION EXCHANGE CHROMATOGRAPHY
MAN
MUSCLES
PEPTIDES
PH VALUE
PHOSPHATES
PLACENTA
RABBITS
SOLVENT EXTRACTION
STOICHIOMETRY
STRUCTURAL CHEMICAL ANALYSIS
ALDEHYDES
ANIMALS
AZOLES
CARBOHYDRATES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DATA
DECOMPOSITION
ENZYMES
EXTRACTION
FETAL MEMBRANES
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HEXOSES
HYDROLYSIS
IMIDAZOLES
INFORMATION
KINETICS
LABELLED COMPOUNDS
LYSIS
MAMMALS
MEMBRANES
MONOSACCHARIDES
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PRIMATES
PROTEINS
REACTION KINETICS
SACCHARIDES
SEPARATION PROCESSES
SOLVOLYSIS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques