Complete amino acid sequence of branched-chain amino acid aminotransferase (transaminase B) of Salmonella typhimurium, identification of the coenzyme-binding site and sequence comparison analysis
The complete amino acid sequence of the subunit of branched-chain amino acid aminotransferase of Salmonella typhimurium was determined by automated Edman degradation of peptide fragments generated by chemical and enzymatic digestion of S-carboxymethylated and S-pyridylethylated transaminase B. Peptide fragments of transaminase B were generated by treatment of the enzyme with trypsin, Staphylococcus aureus V8 protease, endoproteinase Lys-C, and cyanogen bromide. Protocols were developed for separation of the peptide fragments by reverse-phase high performance liquid chromatography (HPLC), ion-exchange HPLC, and SDS-urea gel electrophoresis. The enzyme subunit contains 308 amino acid residues and has a molecular weight of 33,920 daltons. The coenzyme-binding site was determined by treatment of the enzyme, containing bound pyridoxal 5-phosphate, with tritiated sodium borohydride prior to trypsin digestion. Monitoring radioactivity incorporation and peptide map comparisons with an apoenzyme tryptic digest, allowed identification of the pyridoxylated-peptide which was isolated by reverse-phase HPLC and sequenced. The coenzyme-binding site is a lysyl residue at position 159. Some peptides were further characterized by fast atom bombardment mass spectrometry.
- Research Organization:
- North Carolina State Univ., Raleigh, NC (USA)
- OSTI ID:
- 5607968
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
AMINOTRANSFERASES
BACTERIA
CELL CONSTITUENTS
CHEMICAL COMPOSITION
CHROMATOGRAPHY
COENZYMES
ELECTROPHORESIS
ENZYMES
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
LIQUID COLUMN CHROMATOGRAPHY
MASS SPECTROSCOPY
MEMBRANE PROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NITROGEN TRANSFERASES
ORGANIC COMPOUNDS
PEPTIDES
PLASMIDS
PROTEINS
RECEPTORS
SALMONELLA
SALMONELLA TYPHIMURIUM
SEPARATION PROCESSES
SPECTROSCOPY
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS