electron may be donated by NADH via cytochrome b5 reductase and cytochrome b5. This alternative source of the second electron step is still controversial.
The complex then rearranges with insertion of one atom of oxygen into the substrate to yield the
product. The mechanism is obscure but seems to involve oxygen in an activated form. The other atom of oxygen is reduced to water, the other product. It has been suggested that the hydroxylation of
hydrocarbons by cytochromes P-450 may be a two-step mechanism involving a radical intermediate formed by removal of a hydrogen atom from the substrate and then transference to the oxygen bound to the iron. The hydroxyl may then react with the substrate radical to produce hydroxylated substrate. Under certain circumstances cytochromes P-450 produces hydrogen peroxide. This seems to be when the cycle becomes uncoupled and the oxygenated P-450 complex breaks down differently to give the oxidized cytochrome-substrate complex and hydrogen peroxide.
Cytochromes P-450 may be found in other organelles as well as the SER including the rough
endoplasmic reticulum and nuclear membrane. In the adrenal gland it is also found in the mitochondria, although here adrenodoxin and adrenodoxin reductase are additional requirements in the overall system. Although the liver has the highest concentration of the enzyme, cytochromes P-450 are found in most, if not all, tissues.
The cytochrome P-450 system is a remarkable enzyme system because it consists of so many (iso) enzymes or isoforms (probably at least 40 in man) and results from a gene family represented in all phyla and very many diverse species. More than 300 cDNAs have been cloned and more than 60
different types of chemical reaction are catalysed. This multiplicity of isoforms accounts for the diversity of the reactions catalysed and the substrates accommodated. The forms or isoenzymes can be separated using chromatographic and electrophoretic techniques and the DNA sequences determined using
sophisticated molecular biological techniques. These isoenzymes may vary in distribution both within the cell and in the tissues of the whole organism. The proportions of the isoenzymes in any given tissue may change as a result of treatment with various compounds as described in the next chapter.
There are now a considerable number of isoenzymes which have been identified and some indication of the particular substrates and their characteristics is emerging. Those involved in the metabolism of xenobiotics can be seen in table 4.3. The different isoenzymes are coded for by distinct genes and the nomenclature used in this table is the current internationally accepted standard. This groups enzymes into gene families based on primary amino acid sequence resulting from sequencing of cytochromes P- 450 proteins, cDNAs and genes (Nebert et al., 1991). Prior to the introduction of this nomenclature a confusing array of names was in use based on substrates or inducers and this may still be encountered. Thus the cytochromes P-450 gene superfamily currently consists of 27 gene families.
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TABLE 4.3 Characteristics of cytochromes P-450 families 1–4
Isozyme Substrate examples Reactions
CYP1A1 benzo[a]pyrene hydroxylation
7-ethoxyresorufin O-de-ethylation
1A2 acetylaminofluorene N-hydroxylation
phenacetin O-de-ethylation
CYP2A1 testosterone 7α-hydroxylation
2A2 testosterone 15α-hydroxylation
2A3
CYP2B1 hexobarbital hydroxylation
7-pentoxyresorufin O-de-ethylation
2B2 7-pentoxyresorufin O-de-ethylation
7,12-dimethylbenzanthracene 12-methyl-hydroxylation
CYP2C S-mephenytoin hydroxylation
CYP2D debrisoquine alicyclic hydroxylation
CYP2E1 p-nitrophenol hydroxylation
aniline hydroxylation
CYP3A ethylmorphine N-demethylation
aminopyrine N-demethylation
CYP4A1 lauric acid ω-hydroxylation
lauric acid ω-l-hydroxylation
It should be noted that this is not an exhaustive list and that in different species different numbers of isozymes exist. It serves solely to illustrate the multiplicity of the forms of cytochrome P-450 generally involved with xenobiotic metabolism and the differences and similarities between them.
For a more detailed discussion see Nebert, D.W. and Gonzales, F. (1990) The P-450 gene superfamily. In
Frontiers of Biotransformation, Vol. 2, Principles, Mechanisms and Biological Consequences of Induction, edited
by K.Ruckpaul and H.Rein (London: Taylor & Francis), or other references in the Bibliography.
The various proteins and the genes coding for them are designated by CYP and CYP respectively with the families indicated by Arabic numerals.
There are three main gene families important in xenobiotic metabolism: CYP1, CYP2 and CYP3 and
CYP4 is involved in fatty acid metabolism. However, the latter may be important for some xenobiotics
which have suitable carboxylic acids as part of the structure. The genes in these four families code for primarily hepatic, microsomal enzymes.
Within these families there is one subfamily, i.e. CYP1A, CYP3A, CYP4A, except CYP2 which has five subfamilies A, B, C, D and E. These may be further divided into genes coding for single distinct enzyme proteins such as CYP1A1 and CYP1A2. Subfamilies are designated by capital letters and proteins and genes within those by Arabic numerals.
There may also be allelic variants giving rise to different proteins.
The four families CYP17, CYP19, CYP20 and CYP22 code for P-450s involved in steroid biosynthesis and found mainly in extrahepatic tissues. The mitochondrial P-450 is CYP11. The enzyme proteins CYP1A1, CYP1A2 and CYP2EI seem to be highly conserved and similar in all species. Humans have at
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enzymes. There are also other forms of P-450 to be found in insects, yeast and bacteria. For further details on this topic the reader is referred to the Bibliography.
As already indicated, however, there is overlap between some of the P-450s in terms of substrates and types of reaction catalysed. These different isoenzymes may be separated on the basis of certain criteria. Thus they may have different monomeric molecular weights, the carbon monoxide difference spectra may show different maxima, the amino acid composition and terminal sequences may be different, substrate specificities may be different and they may be distinguished by specific antibodies. The
importance of these different isoenzymes is that they may catalyse different biotransformations and this may be crucial to the toxicity of the compound in question. Variations in the proportions and presence of particular isoenzymes may underly differences in metabolism due to species, sex, age, nutritional status and interindividual variability. The comparison and identification of particular forms (orthologues) of cytochrome P-450 between species has proved to be difficult in some cases, however. The presence or absence of a particular isoenzyme may be the cause of toxicity in one organ or tissue. The change in the proportion of isoenzymes caused by exposure to substances in the environment, or drugs may explain changes in toxicity or other biological activity attributed to the compound of interest. These questions will be considered in greater detail in the following chapter.
One important feature of the cytochrome P-450 enzyme system is its broad and overlapping substrate specificity which reflects the enormous variety of chemicals which may be potential substrates.
Furthermore one substrate may be metabolized to more than one product by different forms of
cytochrome P-450. For example the drug propranolol can be metabolized by CYP2D6 and CYP2C19 to 4-hydroxypropranolol and naphthoxylacetic acid respectively. Sometimes the same form of cytochrome P-450 may metabolize one drug to more than one product. For example the drug methoxyphenamine can be metabolized by CYP2D6 either by O-demethylation or hydroxylation on the 5 position. Despite this lack of substrate specificity however, the enzyme may display significant stereoselectivity with chiral substrates (see below). There is an enormous variety of substrates for cytochromes P-450, and the only seemingly common factor is a degree of lipophilicity. Indeed there is a correlation between the
metabolism of xenobiotics by microsomes and the lipophilicity of the compound. This is not surprising in that if the purpose of metabolism is to increase the water solubility of a foreign compound and hence its excretion, the compounds most needing this biotransformation are the lipophilic compounds.
Furthermore, the lack of substrate specificity requires some control if many vital endogenous molecules are not to be wastefully metabolized. This control is exercised by the lipoidal character of the enzyme complex which effectively excludes many endogenous molecules. Cytochromes P-450 may also be involved in the metabolism of endogenous compounds, particularly in some tissues where the
appropriate isoenzyme is located, but these substrates again tend to be lipophilic. For example in the kidney fatty acids are substrates, undergoing ω-1 hydroxylation, and prostaglandins also undergo this type of hydroxylation. In the adrenal cortex, steroids are hydroxylated by a mitochondrial cytochromes P-450.
Although in the majority of cases, cytochromes P-450 catalyse oxidation reactions, under certain circumstances the enzyme may catalyse other types of reaction such as reduction.