Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes.
Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.
Inouye
K
K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan. inouye@kais.kyoto-u.ac.jp
Mizokawa
T
T
Saito
A
A
Tonomura
B
B
Ohkawa
H
H
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D003374:Coumarins / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics / Q000378:metabolism*; D005990:Glycerol; D007700:Kinetics; D000432:Methanol; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature
D003374:Coumarins; C017299:7-ethoxycoumarin; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1; D005990:Glycerol; D010100:Oxygen; D000432:Methanol