Identification of Lys116 as the target of N-ethylmaleimide inactivation of ferredoxin:NADP+ oxidoreductase.
Oxidized ferredoxin:NADP+ oxidoreductase (FNR) was slowly and irreversibly inactivated by N-ethylmaleimide. Complete protection against inactivation was afforded by saturating concentrations of NADP+. In the presence of NADPH, a rapid inhibition of the enzyme ensued; however, this inhibition was found to be reversible. In the tryptic map of the flavoprotein, modified with N-ethyl[2,3-14C]maleimide in oxidizing conditions, a unique radioactive peptide was found. Its sequence comprised residues 110-117 of the enzyme: Lys116 was shown to be the residue alkylated by N-ethylmaleimide. It is noteworthy that the same residue of FNR was found to be modified by 5-dimethylaminoaphthalene-1-sulfonyl(dansyl) chloride at the putative NADP(H)-binding site [Cidaria, D., Biondi, P. A., Zanetti, G. & Ronchi, S. (1985) Eur. J. Biochem. 146, 295-299]. Furthermore, the data reported here demonstrate that the sulfhydryl groups of FNR are not involved in enzyme inactivation by N-ethylmaleimide.
Aliverti
A
A
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Gadda
G
G
Ronchi
S
S
Zanetti
G
G
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000737:chemistry; D005033:Ethylmaleimide / Q000494:pharmacology*; D006736:Horses; D008239:Lysine / Q000378:metabolism*; D009249:NADP / Q000032:analysis; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010449:Peptide Mapping; D014357:Trypsin
D000596:Amino Acids; D003619:Dansyl Compounds; D009249:NADP; D010088:Oxidoreductases; C028878:ferredoxin-NAD+ reductase; D014357:Trypsin; D008239:Lysine; D005033:Ethylmaleimide