Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH/sub 2/-terminal amino acid sequence

Journal Article · Sat Jan 01 00:00:00 EST 1977 · Arch. Biochem. Biophys.; (United States)

DOI:https://doi.org/10.1016/0003-9861(77)90236-3· OSTI ID:6550051

Hsu, R; Singer, S J; Keim, P; Deuel, T F; Heinrikson, R L

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxymethylated protein revealed a single NH/sub 2/-terminal amino acid sequence: H/sub 2/N-Ala-Lys-Tyr-Thr-Arg/sup 5/-Glu-Asp-Ile-Gln-Lys/sup 10/-Leu-Val-Ser-Glu-Ser/sup 13/-CM-Cys-Val-Thr-Tyr-Ile/sup 20/-Ser-Leu-Gly-Phe-Ser/sup 25/-Asn-Ser-Leu-Gly--. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH/sub 2/ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5'-dithiobis(2-nitrobenzoic acid).

• Research Organization: Univ. of Chicago
• OSTI ID: 6550051
• Journal Information: Arch. Biochem. Biophys.; (United States), Vol. 178
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BIOCHEMICAL REACTION KINETICS
BACILLUS SUBTILIS
LIGASES
MOLECULAR STRUCTURE
PROTEINS
CYSTEINE
MOLECULAR WEIGHT
PURIFICATION
RESIDUES
BACILLUS
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
KINETICS
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
REACTION KINETICS
THIOLS
550200* - Biochemistry