A lysyl residue at the NADP binding site of ferredoxin-NADP reductase.
Dansyl chloride, at low molar ratio, inactivates ferredoxin-NADP reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1). The complete protection afforded either by NADP or NADPH suggests a direct involvement of the active site. Experiments with [Me-14C] dansyl chloride showed that about 1.5 residues per flavin were dansylated: by differential labelling experiments using NADP, it has been proved that enzyme inactivation is due to dansylation of one residue. The group modified has been identified as the epsilon-amino group of a lysine. The pH-inactivation profile indicates that this essential group has an apparent pKa of 8.7. The dansylated flavoprotein seems to maintain its native conformation; it shows a fluorescent chromophore with a peak at 335 nm. The modified enzyme has lost the capacity to form a complex with NADP, nevertheless it interacts normally with ferredoxin. It is concluded that the loss of catalytic activity which parallels the dansylation of a lysyl residue occurs because this residue is essential for the binding of the pyridine nucleotide substrate. Protection experiments with a series of coenzyme analogs further indicate that this lysyl residue interacts, most likely, with the 2'-phosphate moiety of NADP(H).
D000249:Adenosine Monophosphate / Q000494:pharmacology; D001665:Binding Sites; D003619:Dansyl Compounds / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005288:Ferredoxins / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D008239:Lysine; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry
D003619:Dansyl Compounds; D005288:Ferredoxins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D008239:Lysine