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), Journal Name: J. Biol. Chem.; (United States) Vol. 259:5; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
BACTERIA
CARBOHYDRATES
CARBON 14 COMPOUNDS
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLASE
CHROMATOGRAPHY
COVALENCE
DATA
ENZYME ACTIVITY
ENZYMES
EXPERIMENTAL DATA
FOOD
INFORMATION
ISOTOPE APPLICATIONS
KETONES
LABELLED COMPOUNDS
LABELLING
LYASES
MICROORGANISMS
MONOSACCHARIDES
NUMERICAL DATA
ORGANIC COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
OXYGENASES
PENTOSES
PEPTIDES
PHOSPHATES
PHOSPHORUS COMPOUNDS
PLANTS
PROTEINS
PURIFICATION
RHODOSPIRILLUM
RIBULOSE
SACCHARIDES
SEPARATION PROCESSES
SPINACH
TRACER TECHNIQUES
VEGETABLES