Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase.
To identify amino acids of cytochrome P450d (P450d) which participate in the interaction with NADPH-cytochrome P450 reductase, we changed conserved ionic amino acids of P450d to others by site-directed mutagenesis. Turnover numbers (0.032-0.008 min-1) of purified mutants Lys94-Glu, Lys99-Glu, Lys105-Glu, Lys440-Glu, Lys453-Glu, Arg455-Glu, and Lys463-Glu toward 7-ethoxycoumarin were much lower than that (0.380 min-1) of the wild type at 25 degrees C. Reduction rates (less than 0.054 s-1) of the heme of all mutants (0.1 microM) in the presence of NADPH and the reductase (0.3 microM) were much lower than that (5.9 s-1) of the wild type. Furthermore, a turnover number (0.042 min-1) of a microsomal triple mutant (Arg135-Leu + Arg136-Leu + Arg137-Leu) of a conserved Arg cluster was much lower than that (0.674 min-1) of the wild type at 37 degrees C. Thus, we suggest that Lys94, Lys99, Lys105, Lys440, Lys453, Arg455, Lys463, and perhaps the Arg cluster Arg135-Arg136-Arg137 of P450d will participate in the intermolecular electron transfer process by forming ionic bridges between the two proteins and/or by orienting appropriate geometry for electron transfer on the interfacial surface between the two proteins.
Shimizu
T
T
Chemical Research Institute of Non-aqueous Solutions, Tohoku University, Katahira, Sendai, Japan.
Tateishi
T
T
Hatano
M
M
Fujii-Kuriyama
Y
Y
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment
D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D009251:NADPH-Ferrihemoprotein Reductase; D008239:Lysine