2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum: determination of reaction parameters and characterization of an active site peptide
Reductive amination of ribulose-P/sub 2/ with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido)anilino-2-deoxypentitol 1.5-bisphosphate) which were competitive inhibitors of the carboxylase with K/sub i/ values of 705 and 104 ..mu..M, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO/sub 2/ and Mg/sup 2 +/, however, resulted in an irreversible, time-dependent loss of activity, with a K/sub inact/ of 125 ..mu..M and a minimal half-time of 7.3 min. Covalent incorporation of (/sup 14/C)reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of (/sup 14/C)reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. 60 references, 7 figures, tables.
- Research Organization:
- Oak Ridge National Lab., TN
- DOE Contract Number:
- W-7405-ENG-26
- OSTI ID:
- 5887156
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 259:5
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CARBOXYLASE
ENZYME ACTIVITY
LABELLING
OXYGENASES
PEPTIDES
PURIFICATION
AFFINITY
CARBON 14 COMPOUNDS
CHROMATOGRAPHY
COVALENCE
EXPERIMENTAL DATA
PHOSPHATES
RHODOSPIRILLUM
RIBULOSE
SPINACH
TRACER TECHNIQUES
BACTERIA
CARBOHYDRATES
CARBON-CARBON LYASES
CARBOXY-LYASES
DATA
ENZYMES
FOOD
INFORMATION
ISOTOPE APPLICATIONS
KETONES
LABELLED COMPOUNDS
LYASES
MICROORGANISMS
MONOSACCHARIDES
NUMERICAL DATA
ORGANIC COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PENTOSES
PHOSPHORUS COMPOUNDS
PLANTS
PROTEINS
SACCHARIDES
SEPARATION PROCESSES
VEGETABLES
550201* - Biochemistry- Tracer Techniques