Isolation and sequencing of an active-site peptide from spinach ferredoxin-NADP+ oxidoreductase after affinity labeling with periodate-oxidized NADP+.
Spinach ferredoxin-NADP+ oxidoreductase was inactivated by treatment with 2',3'-dialdehyde NADP+ (periodate-oxidized NADP+), which selectively modifies a lysine residue at the nucleotide-binding domain of the enzyme. The identity of the derivatized residue was ascertained by thin-layer chromatography of the protein hydrolysate. Reductase that had been labeled with periodate-oxidized NADP+ and NaB3H4 was treated with trypsin, and samples of the tryptic digest were subjected to reverse-phase high-performance liquid chromatography. The radioactivity profiles showed modification of one specific peptide. The primary structure of this peptide was found to be Gly-Glu-Lys*-Met-Tyr-Ile-Gln-Thr-Arg, where Lys* represents the derivatized lysine. The sequence obtained corresponds to residues 242-250 in the primary structure of spinach ferredoxin-NADP+ reductase recently reported [Karplus et al. (1984) Biochemistry 23, 6576-6583].
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology*
D000345:Affinity Labels; D000596:Amino Acids; D010446:Peptide Fragments; D009249:NADP; C038805:2',3'-dialdehyde NADP; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases