Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate

Journal Article · Sat Jan 01 00:00:00 EST 1983 · Biochemistry; (United States)

OSTI ID:6000984

Fraij, B; Hartman, F C

2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. The enzyme has now been inactivated with a /sup 14/C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln-Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser-Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site. 31 references, 4 figures, 3 tables.

• Research Organization: Oak Ridge National Lab., TN
• DOE Contract Number: W-7405-ENG-26
• OSTI ID: 6000984
• Journal Information: Biochemistry; (United States), Vol. 22:6
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CARBOXYLASE
ENZYME ACTIVITY
OXYGENASES
PEPTIDES
PURIFICATION
STRUCTURAL CHEMICAL ANALYSIS
AFFINITY
AMINO ACID SEQUENCE
BIOCHEMISTRY
CARBON 14 COMPOUNDS
EXPERIMENTAL DATA
PHOSPHORIC ACID ESTERS
RHODOSPIRILLUM
SPINACH
TRACER TECHNIQUES
BACTERIA
CHEMISTRY
DATA
ENZYMES
ESTERS
FOOD
INFORMATION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIGASES
MICROORGANISMS
MOLECULAR STRUCTURE
NUMERICAL DATA
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXIDOREDUCTASES
PLANTS
PROTEINS
VEGETABLES
550201* - Biochemistry- Tracer Techniques