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

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°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.

Introduction

Hepatic microsomal P-450s play an important role in the metabolism of lipophilic compounds such as drugs and xenobiotics. Of these microsomal P-450 isoforms, P-4501A1 appears to be one of the most extensively studied, since induction of P-4501A1 by polycyclic aromatic hydrocarbons such as dioxins and arylhydrocarbon hydroxylase activity of P-4501A1 are closely related with mutation and cancer [1], [2], [3], [4]. Thus, the elucidation of the induction mechanism and enzymatic properties of P-4501A1 seems quite significant for human health science.

Enzymatic analysis of single P-450 isoform using mammalian microsomes seems very difficult since the microsomes contain multiple P-450 isoforms. Although specific compounds such as 3-methylcholanthrene induce the expression of P-4501A1, the microsomes also contain 3-methylcholanthrene-inducible P-4501A2 [5] and small amounts of other P-450 isoforms [6]. On the other hand, reconstituted systems consisting of the purified P-4501A1 and NADPH-P-450 reductase with synthetic phospholipids and/or detergent [7], [8], [9], [10] appear to have a risk to show significantly different results from those in physiological conditions. As reported previously [11], [12], [13], [14], the recombinant yeast microsomal membrane containing P-4501A1 seems very useful for the kinetic analysis of P-4501A1. The recombinant yeast microsomal membrane would be considered as a well-organized reconstituted membrane containing only P-4501A1 as P-450 isoforms. Although the recombinant yeast microsomal membrane contains yeast NADPH-P-450 reductase instead of the mammalian NADPH-P-450 reductase, the similarity of both reductases as an electron donor to mammalian microsomal P-450s has been confirmed as described previously [15], [16], [17]. In addition, the great advantage of the yeast expression system is an easy preparation of enough amounts of the recombinant yeast microsomes for kinetic studies as compared with mammalian cell expression systems.

In this study, we will discuss the biphasic kinetic behaviors of P-4501A1-dependent monooxygenations, and the effect of glycerol that was used as a stabilizer of P-450 in microsomes [18], and also the effect of methanol which was used as a solvent of the substrate 7-ethoxycoumarin.