Multi-step oxidations catalyzed by cytochrome P450 enzymes: Processive vs. distributive kinetics and the issue of carbonyl oxidation in chemical mechanisms

Abstract

Catalysis of sequential oxidation reactions is not unusual in cytochrome P450 (P450) reactions, not only in steroid metabolism but also with many xenobiotics. One issue is how processive/distributive these reactions are, i.e., how much do the "intermediate" products dissociate. Our work with human P450s 2E1, 2A6, and 19A1 on this subject has revealed a mixture of systems, surprisingly with a more distributive mechanism with an endogenous substrate (P450 19A1) than for some xenobiotics (P450s 2E1, 2A6). One aspect of this research involves carbonyl intermediates, and the choice of catalytic mechanism is linked to the hydration state of the aldehyde. The non-enzymatic rates of hydration and dehydration of carbonyls are not rapid and whether P450s catalyze the reversible hydration is unknown. If carbonyl hydration and dehydration are slow, the mechanism may be set by the carbonyl hydration status.

Fig. 1

Multi-step oxidations of steroids catalyzed by human P450 enzymes [5].

Fig. 2

Stepwise oxidation of ethanol to acetic acid by human P450 2E1. Three possible mechanisms are shown for the oxidation of acetaldehyde to acetic acid in parts B-D.

Fig. 3

Pre-steady-state kinetic burst observed for oxidation of ethanol to acetaldehyde with P450 2E1 [20].

Fig. 4

A kinetic mechanism for the oxidation of ethanol to acetic acid by P450 2E1 [21]. Rate constants are shown in units of s^-1 and M^-1.

Fig. 5

Oxidation of DMN and DEN to aldehydes and carboxylic acids by P450 2A6.

Fig. 6

Pulse chase experiments with P450 2A6 [28]. Reactions were initiated with labeled nitrosamines. After the indicated time, unlabeled aldehyde (1.5 or 2 mM) was added and the reactions proceeded 20 min, at which time the labeled carboxylic acids were quantified as described in the original reference. (A) DMN. (B) DEN.

Fig. 7

A general scheme of oxidation of dialkylnitrosamines [28]. The indicated rate constants are for non-enzymatic hydration and dehydration of the aldehydes [40,41] and for hydrolysis of the α-hydroxy nitrosamine [42].

Fig. 8

A kinetic mechanism for oxidation of dialkylnitrosamines by human P450 2A6 [28].

Fig. 9

An alternate mechanism for the oxidation of alkylnitrosamines to carboxylic acids, with the intermediate α-hydroxy nitrosamine serving as a substrate for the second reaction. The half-lives of the unstable products are indicated (at pH 7.4, 37 °C)

Fig. 10

Pulse chase experiments with human P450 19A1. The reaction was initiated with [¹⁴ C]androstenedione, and the indicated compounds were added after 1 min, followed by further incubation for 19 min and analysis of radiolabeled estrone [34].

Fig. 11

Fitting of a single-turnover P450 19A1 androstenedione reaction using the model and rate constants of reference [34] to a minimal kinetic mechanism.

Fig. 12

Alternate chemical mechanisms for the third step of the P450 19A1 oxidation.

Fig. 13

The hydration issue with the aldehyde in the third step of the P450 19A1 reaction, which is general for multi-step P450 oxidations involving carbonyl intermediates.