Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes

Biochim Biophys Acta. 2000 Sep 29;1481(2):265-72. doi: 10.1016/s0167-4838(00)00135-7.

Abstract

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.

MeSH terms

  • 7-Alkoxycoumarin O-Dealkylase / metabolism
  • Animals
  • Coumarins / metabolism
  • Cytochrome P-450 CYP1A1 / genetics
  • Cytochrome P-450 CYP1A1 / metabolism*
  • Glycerol
  • Kinetics
  • Methanol
  • Microsomes / enzymology
  • Microsomes / metabolism*
  • Microsomes, Liver / enzymology
  • Oxygen / metabolism*
  • Rats
  • Recombination, Genetic
  • Saccharomyces cerevisiae / genetics
  • Temperature

Substances

  • Coumarins
  • 7-ethoxycoumarin
  • 7-Alkoxycoumarin O-Dealkylase
  • Cytochrome P-450 CYP1A1
  • Glycerol
  • Oxygen
  • Methanol