Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin.
Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.
Furuya
H
H
Chemical Research Institute of Non-Aqueous Solutions, Tohoku University, Sendai, Japan.
Shimizu
T
T
Hirano
K
K
Hatano
M
M
Fujii-Kuriyama
Y
Y
Raag
R
R
Poulos
T L
TL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001589:Benzphetamine / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D008099:Liver / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010627:Phenethylamines / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D013728:Terminator Regions, Genetic
D003374:Coumarins; D010627:Phenethylamines; D001589:Benzphetamine; C017299:7-ethoxycoumarin; D003577:Cytochrome P-450 Enzyme System