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Reexamination of the binding site for pyridoxal 5'-phosphate in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Journal Article · · Biochemistry; (United States)
DOI:https://doi.org/10.1021/bi00535a043· OSTI ID:7071826
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The high specificity of pyridoxal 5'-phosphate (PLP) for an essential lysyl residue of ribulosebisphosphate carboxylase/oxygenase was confirmed, but half-of-sites reactivity was not observed. Subsequent to reduction with (/sup 3/H)borohydride and tryptic digestion of the enzyme inactivated by PLP, the sole labeled peptide was purified by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. The peptide, recovered in good yield, appeared essentially homogeneous by amino acid analysis, peptide mapping, and sequencing. Automated Edman degradation established the peptide's sequence as Val-Leu-Gly-Arg-Pro-Glu-Val-Asp-Gly-Gly-Leu-Val-Val-Gly-Thr-Ile-Ile-(PLP)Lys-Pro-Lys instead of Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP)Lys-Gly-Thr-Leu-Val-Ile-Lys. The sequence -Ile-Lys-Pro-Lys- in the former is identical with that encompassing Lys-175 in the carboxylase/oxygenase from spinach, which reacts preferentially with PLP and two other affinity labels. This finding of homology greatly strengthens the supposition that Lys-175 in the spinach enzyme and the corresponding lysyl residue in the Rhodospirillum rubrum enzyme are active-site residues and furthermore increases the likelihood of their functionality in catalysis. (JMT)

Research Organization:
Oak Ridge National Lab., TN
DOE Contract Number:
W-7405-ENG-26
OSTI ID:
7071826
Journal Information:
Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 21:6; ISSN BICHA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
AMINO ACIDS
AZINES
BACTERIA
CARBOHYDRATES
CARBOXYLASE
CARBOXYLIC ACIDS
CHEMICAL BONDS
CHROMATOGRAPHY
ENZYMES
HETEROCYCLIC COMPOUNDS
INACTIVATION
ISOTOPE APPLICATIONS
KETONES
LABELLED COMPOUNDS
LIGASES
MICROORGANISMS
MONOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
OXYGENASES
PENTOSES
PEPTIDES
PHOSPHATES
PHOSPHORUS COMPOUNDS
PROTEINS
PURIFICATION
PYRIDINES
PYRIDOXAL
RHODOSPIRILLUM
RIBULOSE
SACCHARIDES
SEPARATION PROCESSES
STRUCTURAL CHEMICAL ANALYSIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS