CYPs belong to a group of enzymes which all have similar core structures and modes of operation. Although these enzymes were discovered in 1958, vast amounts of research have not yet revealed all there is to know of their structure and function. Their importance to us is underlined by their key role in more than 75 per cent of all drug biotransformations. In all living things, over 7,700 individual CYPs have been described so far, although humans make do with just 57; of these, only 15 metabolize drugs and other xenobiotics. Many of the other CYPs are poorly understood in terms of their physiological function and regulation and have been termed ‘orphan’ CYPs.
In principle, to understand how CYPs operate, it is first necessary to discover their detailed structure. Currently, every CYP from any source is classified according to its amino acid sequence homology, that is, if two CYPs have 40 per cent of the full length of their amino acid structure in common they are assumed to belong to the same ‘family’. To date, more than 780 CYP families have been found in nature in total, but only 18 have been identified in humans. The families are numbered, such as CYP1, CYP2, CYP3, etc. Subfamilies are identified as having 55 per cent sequence homology; these are identified by using a letter and there are often several subfamilies in each family. So you might see CYP1A, CYP2A, CYP2B, CYP2C, etc. Regarding the individual CYP enzymes themselves, these ‘Isoforms’ originate from alleles, or slightly different versions of the same gene. They are given numbers within the subfamily, such as CYP1A1 or CYP1A2 and these isoforms have 97 per cent of their general sequences in common. From a practical point of view, differences in the binding site amino acid sequences of the isoforms rather than the full length structure are likely to be more relevant in terms of which specific molecules these enzymes can actually metabolize. With CYP2D6, it is known that change in just one amino acid residue in the binding site is crucial in substrate binding. The amino acid sequences of many bacterial, yeast and mammalian enzymes are now well known and this has underlined some large differences, as well as surprising similarities between the structures of our own CYPs and those of animals, eukaryotes and bacteria. Interestingly, the same metabolite of a given substance will be made by different CYPs across species. Ideally, it’s often easiest to understand how machines work by watching cutaway models, like the ones seen of engines at motor shows. With enzymes in living systems, options are much more limited and the most practical method is to ‘catch it in the act’, that is, to crystallize it when it is bound to a substrate. Then, the technique of X-ray crystallography can be used to explore and map the contours and features of the enzyme. Some CYPs are water-soluble, such as P450cam, which was crystallized relatively early on, affording the opportunity to study it in detail. As mammalian CYPs function in a lipid environment, this renders crystallization exceedingly difficult. However, by making certain minor external structural modifications to improve water solubility, in 2000 a rabbit CYP (2C5) was crystallized, followed in 2003 by the crystallization of some of the most important human CYPs, CYP2C9, and CYP3A4. Since then, other human drug metabolizing CYPs have also been crystallized, such as CYP2D6, CYP2C8 and CYP2A6.
However, in some ways, using crystallography in this context is like studying how an animal runs or how a machine operates through a series of ‘freeze frames’, rather than being able to watch the process operating in real time in a ‘natural’ environment. Indeed, with any research technique, findings are influenced by the limitations of that technique and X-ray crystallography requires the subjection of the CYP isoforms to extremely unphysiological conditions. In some cases, such as CYP2C9, several crystal structures exist, each capturing the isoform binding a different substrate. Nonetheless, great progress has been made in our understanding of external CYP structure, including details of the access and egress pathways for substrates and products, as well as the dimensions of the inner structure such as the active site. The many basic structural similarities between the CYPs have also been revealed using crystallography. As mentioned above, because crystallography is a ‘freeze frame’ technique, it has been much harder to determine how CYPs actually operate catalytically and it has also been particularly difficult to determine the key to their flexibility; that is, how they can apparently recognize and bind so many disparate groups of substrates. These range from large molecules such as the immunosuppressant cyclosporine, down to relatively small entities such as ethanol and acetone. The advent of many in silico, or computer-based techniques such as molecular dynamics, have allowed researchers to understand in much more detail how the protein structures of CYPs unwind and unfold to provide that remarkable degree of flexibility during substrate binding.
Although CYPs in general are capable of metabolizing almost any chemical structure, they do have a number of features in common:
• Most mammalian CYPs exist in a so-called ‘lipid microenvironment’. This means that the CYP is partially embedded in the lipophilic membrane of the SER and their access channels are actually positioned inside the membrane ready to receive lipophilic substrates, rather like having an underwater entrance to a tropical island cave.
• CYPs feature a haem group in their active site which contains iron, which is a crucial and highly conserved part of their structures. This area is quite rigid, but it is surrounded by much more flexible complex binding areas.
• All CYPs contain at least one binding area in their active site, which is the main source of their variation and their ability to metabolize a particular group of chemicals.
• To catalyze substrate oxidations and reductions, CYPs exploit the ability of a metal, iron, to gain or lose electrons, rather like a rechargeable battery in a cordless drill. (Figure 3.1).
• They all have closely associated ‘REDOX partners’, which are P450 oxidoreductase (POR) and cytochrome b5, that supply them with electrons to ‘fuel’ their catalytic activities (Figure 3.1 ).
• They all bind and activate oxygen as part of the process of metabolism.
• They are all capable of reduction reactions that do not require oxygen.
