Probing the role of lysine 116 and lysine 244 in the spinach ferredoxin-NADP+ reductase by site-directed mutagenesis.
Two mutants of the spinach ferredoxin-NADP+ reductase (FNR) were constructed, expressed by using a heterologous expression system previously described (Aliverti, A., Jansen, T., Zanetti, G., Ronchi, S., Herrmann, R. G., and Curti, B. (1990) Eur. J. Biochem. 191, 551-555), and purified to homogeneity. The mutant enzymes FNR-Lys116Gln and FNR-Lys244Gln were similar to the wild-type enzyme in the physicochemical properties measured; however, both enzymes showed a lower activity. Steady-state kinetic analyses and NADP+ binding measurements of the mutant proteins have revealed that the Lys116Gln enzyme had a catalytic efficiency (kcat/KmNADPH) 500-fold lower than that of the wild-type enzyme, and its interaction with NADP+ was greatly impaired. The Lys244Gln enzyme instead had almost the same kcat in the ferricyanide reductase reaction as the wild-type enzyme, while higher values of KmNADPH and Kd for NADP+ were observed. Thus, protein engineering has enabled us to define the role of Lys-116 and Lys-244 in ferredoxin-NADP+ reductase; whereas Lys-244 contributes to the stabilization of the Michaelis complex, the Lys-116 side chain plays an important role also in the further steps of the catalytic cycle, because both Km and kcat are highly affected by the mutation.
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
A
A
Aliverti
T
T
Lübberstedt
G
G
Zanetti
R G
RG
Herrmann
B
B
Curti
D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005287:Ferredoxin-NADP Reductase / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology*
D005287:Ferredoxin-NADP Reductase; D008239:Lysine