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Ernest Hodgson, I Nathan 1. Cherrington, I Richard M. Philpot,z and

Randy L. Rosel

IDepartment of Toxicology
North Carolina State University
Raleigh, North Carolina 27695-7633
2National Institute for Environmental Health Sciences
Research Triangle Park, North Carolina 27709


1.1. Historical

Xenobiotics are metabolized by many enzymes, including a number of isoforms of

cytochrome P450 (P450), the flavin-containing monooxygenases (FMO), prostaglandin
synthetase, alcohol and aldehyde dehydrogenases, molybdenum hydroxy lases, esterases,
as well as a number of transferases, particularly the glutathione S-transferases, the glu-
curonyl transferases and the sulfotransferases.1.2 Both activation and detoxication reac-
tions can be catalyzed by any of these enzymes but P450 is the most important with regard
to activation of toxicants. While many of the reactions carried out by the FMO and P450
are similar there are differences between them. The most significant of these differences
are that FMO does not, so far as is known, carry out oxidations at carbon atoms,3 prefering
soft nucIeophiles as substrates and carrying out oxidations at heteroatoms, such as nitro-
gen, sulfur, selenium and phosphorus, in organic molecules.
Oxygen and NADPH-dependent microsomal oxidations were attributed, in a land-
mark 1963 paper by Estabrook et a\.4 to P450. This attribution has proven to be correct as
P450 isoforms are now known to oxidize a huge array of both xenobiotics and endogenous
metabolites through a large number of different chemical reactions. At the same time,
workers studying the oxidation of N,N-dimethylaniline and other secondary and tertiary
amines determined that another enzyme, differing from P450 in thermal stability and sen-
sitivity to carbon monoxide, was present in hog liver microsomes. 5- 7 This enzyme, termed


E. Arin et al. (eds.), Molecular and Applied Aspects of Oxidative Drug Metabolizing Enzymes
Kluwer Academic / Plenum Publishers, New York 1999

E. Hodgson et al.


~ .,-NADPH

\ +W




Figure J. Catalytic cycle of FMO.

amine oxidize and other names, including "Ziegler's enzyme", was purified from hog liver
and shown to contain flavin-adenine dinucleotide (FAD).8
It was subsequently determined that this "amine oxidase" had a wider range of sub-
strates, and a more appropriate name, the flavin-containing monooxygenase (usually abbre-
viated to FMO)(EC, was adopted. Among organic compounds metabolized by
FMO are those containing nitrogen, sulfur or phosphorus heteroatoms. 9- 13 More recently,
boron and selenium containing compounds have been identified as FMO substrates. 14 . IS

1.2. Cytochrome P450 and FMO--Mechanisms Compared

In mammals, both FMO and P450, particularly the P450 isoforms involved in the
oxidation of xenobiotics, have similar distribution in the endoplasmic reticulum as well as
similar organ distribution, being at high levels in the liver but broadly distributed in such
tissues as the nervous system, lungs, kidney and skin. However, for particular substrates
the relative importance may vary due to differential distribution of isoforms among or-
gans. Both FMO and P450 require NADPH and O2 but only in the case ofP450 is a reduc-
tase required to transfer electrons from NADPH to the mono oxygenase. The biggest
difference between FMO and P450 lies in the mechanism of oxidation. The FAD pros-
thetic group of FMO first reacts with NADPH and then molecular oxygen to give rise to
the enzyme bound hydroperoxyflavin responsible for the oxidation of suitable substrates.
These initial reactions occur in the absence of substrate and, in the resting state, the en-
zyme exists primarily in the hydroperoxyflavin form. The consequence, in terms of sub-
strate level kinetics, of this series of events (Figure I) is that all substrates, with very few
exceptions, have the same Vmax, although Km may vary. 16
The mechanism by which P450 oxidizes xenobiotic substrates is quite different. The
initial step is the binding of substrate to the oxidized enzyme followed by a one electron
reduction catalyzed by the NADPH-P450 reductase to form a reduced cytochrome-sub-
strate complex. The next several steps include reaction with molecular oxygen to form a
ternary oxygenated complex, followed by a second electron transfer, the electron usually
derived from the P450 reductase but, in some cases, from cytochrome b s to form, eventu-
ally, a complex that breaks down to yield the oxygenated product, water and the oxidized
cytochrome (Figure 2). The consequence of this sequence, relative to substrate level kinet-
ics, is that both Vmax and Km may vary. 16