Effect of mutations of ionic amino acids of cytochrome P450 1A2 on catalytic activities toward 7-ethoxycoumarin and methanol.
Catalytic efficiencies, percentages of rates of product formation per NADPH oxidized, and rates of product formation per O2 consumed of ionic mutants of cytochrome P450 1A2 (P450 1A2) were studied. Efficiencies of Lys99Glu, Lys453Glu, and Arg455Glu mutants for the hydroxylation reaction toward 7-ethoxycoumarin in the reconstituted system were much lower than that of the wild type (less than 17%), which corresponds to lower turnover numbers for these mutants. In contrast, the catalytic efficiencies for the hydroxylation reaction toward methanol of the three mutants were more than 45% that of the wild type in spite of these mutants' lower turnover numbers. Turnover numbers and catalytic efficiencies of Arg137Leu and Lys401 Glu mutants toward both substrates were comparable to those of the wild type. The electron-transfer rate from the reductase to the heme of P450 1A2 was decreased by 30% upon addition of excess methanol, while it was not influenced by addition of excess 7-ethoxycoumarin. The turnover numbers toward both 7-ethoxycoumarin and methanol as well as the rate constant of electron transfer were decreased by 25-40% by raising the concentration of KCl from 0 to 300 mM in the reconstituted system containing 50 mM potassium phosphate buffer. The turnover numbers toward both substrates of the above-mentioned five ionic mutants caused by tert-butyl hydroperoxide in the absence of the reductase and NADPH were comparable to those of the wild type. The effect of phospholipid constituents on the catalytic activity toward 7-ethoxycoumarin of the wild type was also studied.(ABSTRACT TRUNCATED AT 250 WORDS)
Mayuzumi
H
H
Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
Sambongi
C
C
Hiroya
K
K
Shimizu
T
T
Tateishi
T
T
Hatano
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D003374:Coumarins; D010743:Phospholipids; C017299:7-ethoxycoumarin; D000431:Ethanol; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase