DRUG METABOLISM REVIEWS
Vol. 36, No. 2, pp. 159–197, 2004
Cytochrome P450:
What Have We Learned and
What Are the Future Issues?
F. Peter Guengerich*
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt
University School of Medicine, Nashville, Tennessee, USA
ABSTRACT
The cytochrome P450 (P450) field came out of interest in the metabolism of drugs,
carcinogens, and steroids, which remain major focal points. Over the years we have
come to understand the P450 system components, the multiplicity of P450s, and many
aspects of the regulation of the genes and also the catalytic mechanism. Many crystal
structures are now becoming available. The significance of P450 in in vivo
metabolism is appreciated, particularly in the context of pharmacogenetics. Current
scientific issues involve posttranslational modification, gene regulation, component
interactions, structures of P450 complexed with ligands, details of high-valent oxygen
chemistry, the nature and influence of rate-limiting steps, greater details about some
reaction steps, cooperativity, and the relevance of P450 variations to cancer risk. Some
*Correspondence: Prof. F. Peter Guengerich, Department of Biochemistry and Center in Molecular
Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and
Pierce Ave., Nashville, TN 37232-0146, USA; Fax: (615) 322-3141; E-mail: f.guengerich@
vanderbilt.edu.
159
DOI: 10.1081/DMR-120033996
Copyright D 2004 by Marcel Dekker, Inc.
0360-2532 (Print); 1097-9883 (Online)
www.dekker.com
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emerging research areas involve new methods of analysis of ligand interactions, roles
of conformational changes linked to individual reaction steps, functions of orphan
P450s, ‘‘molecular breeding’’ of new P450 functions and enhanced activity, and the
utilization of P450s in chemical synthesis.
Key Words: Cytochrome P450; Ligand binding; Catalytic cycle; Isotope effects;
Polymorphism.
INTRODUCTION
First, I want to express my appreciation to the International Society of Xenobiotics
(ISSX) for the 2003 North American Region Scientific Achievement Award and to the
Xenotech company for its sponsorship. This award was a surprise, because I did not
even know that I had been nominated. I should also point out that this is a
‘‘midcareer’’ award, and I am planning to continue my work for a long time.
This is not an autobiography, but I will make short mention of my background and
thank a few people. Although you might not appreciate it from reading the lay press,
the United States is a tremendous land of opportunity. I was in the first generation of
the Guengerich family born in this country. My grandfather and father had emigrated
from Bavaria, borrowing money for the trip. My father had not gone to high school, but
in retrospect I realize that most of the work ethic and self-reliance that I have were
learned from him on the farm. Any of the hours I spend pale in comparison to my
father’s life; I am very grateful for the opportunities I have had. I was an undergraduate
at the University of Illinois, where I met Professor Harry P. Broquist (‘‘the Chief’’) as
a sophomore. I spent two summers doing research in his lab. After the first summer, I
was hooked on a research career in biochemistry and never looked back. Before I
graduated from Illinois in 1970, Harry Broquist had decided to move to Vanderbilt and
persuaded me to go there for my graduate studies. As I recall, Duke had offered me
more money and would have been a good place to train, too. However, I had really
liked working with the Chief and went with him to Vanderbilt, receiving my Ph.D. in
1973. I will always remember Prof. Broquist for his love of research, his enthusiasm,
and his fairness in dealing with people. He was a proverbial ‘‘nice guy.’’ The Chief
still lives in Nashville, and we keep in touch.
My graduate studies had focused on bioorganic and natural products chemistry, and
I decided that I would try to learn more about enzymology as a postdoctoral student. I
received offers at Berkeley, Cornell, and Michigan, and I decided to go to Profesor
Minor J. (Jud) Coon’s lab at Michigan. I thought Jud would be a good mentor and that
the cytochrome P450 (P450) work might be interesting because it could have practical
applications some day. In retrospect, this was a good choice. I learned the basics of
P450 research and, even more important, I learned a lot from the lab and from Jud,
even though I know I did not always realize it at the time. In looking back, Jud was a
wonderful example as a professor. I think I learned a lot about running a lab,
interacting with others, the importance of writing well, and what role a professor should
have in advancing a field and science in general.
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I do not wake up every morning and say, ‘‘What would the Chief do, or what
would Jud do?’’ before I go to work. You have to develop your own style and be
yourself. I don’t expect people who trained with me to run their lives exactly like I do
(this probably wouldn’t work anyway). You learn from mistakes you see others make
and your own. Nevertheless, there is much to training about principles and
philosophies, and your choices about whom you train with do matter. I was fortunate
to have excellent mentors; Harry Broquist and Jud Coon are two of the finest people I
have known in science.
In 1975, I received an offer to return to Vanderbilt as an Assistant Professor of
Biochemistry. I had considered possibilities in industry, but the market was fairly slow;
in retrospect, I think this was the job I was meant to take. At the ripe old age of 26, I
began setting up my lab. Starting out as an assistant professor is hard; when I see new
faculty arrive in our department, I think back to the early days and wonder how I made
it. As I think about it, I have far fewer qualms about the future now, but am probably
Figure 1. Some former associates of the Guengerich laboratory at the 2003 North American ISSX
meeting in Providence. Back row (from left): Asit Parikh, Dong-Hyun Kim, Imad Hanna, Tetsuya
Kamataki, Heidi Einolf, F. Guengerich, Chenie Bell-Parikh, Grover Paul Miller, Takahiko Baba,
Hiroshi Yamazaki. Front row (from left): Philip Inskeep, Natilie Hosea, Griff Humphreys, Bill
Brian, Pramod Srivastava. Prof. Kamataki only spent a few weeks working in the lab in 1983, but
we taught him what is now known as ‘‘Western’’ (immuno) blotting. A. Parikh and L.C. BellParikh were graduate students; the others were postdoctoral fellows. Attending the ISSX meeting,
but not included here: Lisa Peterson and Dan Liebler. Thanks to Steve Tannenbaum and Natilie
Hosea for the picture.
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working just as hard. I still love to work in the lab myself; however, administrative and
editorial work takes its toll.
Over the years, I have found my job as a professor to be an interesting one. Of
course, I do some formal teaching to both graduate and medical students. The subject
of my research program is open to my own imagination (and the ability to support it).
One of the roles I have particularly enjoyed is the opportunity to train young scientists,
and I am proud that many of them have been successful in industry and in academia in
several parts of the world. A few of the 13 graduate students and 96 postdoctoral
students who have worked with me are shown in Fig. 1. I have also enjoyed the
opportunities I have had to work with industry, particularly the pharmaceutical
industry. I have learned much about how this work is done and gained considerable
appreciation of the issues in making new drugs. Sometimes I see my fellow academics
and government officials taking either adversarial positions or relegating research in
industry to a secondary position and encouraging their best students to avoid it.
Personally, I do not think it is a good idea to have second-rate scientists making the
new medicines I may be taking someday. Industrial research is an integral part of our
society, unless we think we can get by forever on generic drugs.
Having said all this, I think it is becoming harder to be a professor in the sense I
have known it. Grants are not getting any easier to obtain, and I have concerns about
some of the directions I see in the funding institutes. Universities seem to be in a state
of flux regarding priorities. Nevertheless, I am convinced that the academic and
research systems we have in the United States are still the best in the world and will
survive. Most other nations still want to be like us, or at least to publish in our journals.
Having said that, I have relished the opportunity to travel in this career and to have
friends around the world.
Finally, on the occasion of celebrating my thirtieth wedding anniversary as I finish
this article, I want to acknowledge my wife Cheryl and our three children, Phillip,
Laura, and Anna. I have been privileged to have a wonderful family life to go along
with my scientific career.
Now let me get on with some thoughts about science. I believe that it might
be appropriate to discuss where the P450 world came from and what we have learned.
Part of the impetus for this is my belief that we have not done a good job of teaching history to young scientists and establishing the basis of what we are doing now. I
will also discuss what I believe are areas where we still need answers and emerging
areas of research. These are my own views and should only be interpreted as such.
The discussion is probably light on gene regulation and heavy on chemistry due to
my own bias. I am sure that others might disagree with some of my priorities and
thoughts about directions, but this is why scientists communicate and learn from
each other.
P450—WHERE DID THIS FIELD COME FROM?
In considering what questions have been answered and what remain, there is some
merit in asking how the field began and who we are. Knowing who we are has bearing
on the things that interest us and what we will be doing in the future.
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Much of the seminal work in the area was done before I was born, and my views
of the early days are not first hand. Key early discoveries in the area of drug
metabolism go back to the nineteenth century (Bachmann and Bickel, 1986; Conti and
Bickel, 1977; Guengerich, 1997; Williams, 1947); see also the ISSX website: http://
www.issx.org/hisintro.html). P450 research developed from several areas, including the
metabolism of drugs, carcinogens, and steroids (Estabrook, 2003). Some examples of
early work include the metabolism of azo dyes by the Millers (Mueller and Miller,
1948), the hydroxylation of steroids (Dorfman et al., 1939; Ryan, 1958), and the
isolation of oxidized drugs (Axelrod, 1955). These three areas—drugs, carcinogens, and
steroids—still dominate P450 research. The concept of mixed-function oxidation
stoichiometry was not immediately obvious but was aided by the input of biochemists
into the field, and the concepts of Hayaishi and Mason provided the impetus (Hayaishi
et al., 1955; Mason, 1957), along with the subsequent efforts by Sato (Omura and Sato,
1962) and Estabrook et al. (1963). The development of a microbial P450 system by
Gunsalus in the early 1960s (Bradshaw et al., 1959; Katagiri et al., 1968) ultimately
yielded not only the prototype for sophisticated physical studies (Mueller et al., 1995),
but also a continuing interest in P450 processes in bacteria, an area that is surging
today (vide infra).
So the field was started by people interested in several areas of metabolism
relevant to human health, with microbiology and basic biochemistry involved. This is
how the P450 field looked in its early stages, until the 1960s. In a certain sense, the
nature of the interests in the field is still the same as it was then. What has changed are
the general state of the relevant science, the sophistication of the tools, and the
practical applications of the science, particularly in the pharmaceutical industry.
WHAT QUESTIONS HAVE BEEN ADDRESSED ABOUT P450?
Nature of the System Components
Although the classic ‘‘light-reversal’’ inhibition experiments established P450 as
the terminal acceptor in the microsomal P450 system (Estabrook, 2003; Estabrook et al.,
1963), the pathway for electron flow was still unclear. In 1968, the classic experiments
of Lu and Coon (1968) showed that the rabbit liver microsomal system consisted of
NADPH-P450 reductase and P450, with the phospholipid component providing a
milieu for enhanced interaction. With the exception of cytochrome b5 (b5) in some
situations (vide infra), no other components have been added to the microsomal system.
None of the other factors suggested in the literature have proven to be critical for
function. The mitochondrial P450s receive their electrons from the flavoprotein
NADPH-adrenodoxin reductase and adrenodoxin (a ferredoxin) [some microsomal
P450s can move to the mitochondria and also use their pathway (Anandatheerthavarada
et al., 1998)].
Although the number and arrangement of components is largely settled in the
mammalian systems, the nature of the bacterial P450 systems is more diverse and a
number of modes of electron transport are possible (Nakayama et al., 1996; Roberts
et al., 2003). In some bacteria (e.g., we are presently working with Streptomyces sp.),
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there are multiple ferredoxins and flavoproteins to use (Lamb et al., 2002), and the
combinations are still unclear.
Multiplicity of P450s
Until the 1960s, little consideration had been given to the prospect of multiple
P450s. Clearly, the different steroid hydroxylations were known and the adrenodoxin
dependence of some reactions was recognized, but generally little consideration had
been given to the prospect of multiple P450s within the liver endoplasmic reticulum. In
the 1960s, the results of some rat induction studies (both catalytic activity and
spectroscopy) provided evidence for the presence of multiple entities, or at least two
P450s (Hildebrandt et al., 1968; Sladek and Mannering, 1969). This was more or less
the state of the field when I began my P450 work (i.e., there were two P450s, one
being ‘‘P450’’ and the other ‘‘P448’’). Developments in purification technology led to
convincing evidence for the presence of multiple P450s within liver microsomes
(Haugen et al., 1975). With the discovery of more and more P450s, the field actually
became less clear and the prospect of immunoglobulinlike processing was even
considered (Estabrook and Lindenlaub, 1979; Nebert, 1979).
Today the field has settled down and, with the completion of the human genome,
we have 57 human P450 genes (Table 1), plus a few pseudogenes (Nelson, 2003; see
also http://drnelson.utmem.edu/CytochromeP450.html). As other genomes are completed, definite numbers are being obtained in several species, barring a complete
reevaluation of our concepts about signature sequences.
Table 1. Classification of human P450s based on major substrate class.a
Sterols
Xenobiotics
Fatty acids
Eicosanoids
Vitamins
Unknown
1B1
7A1
7B1
8B1
11A1
11B1
11B2
17
19
21A2
27A1
39
46
51
1A1
1A2
2A6
2A13
2B6
2C8
2C9
2C18
2C19
2D6
2E1
2F1
3A4
3A5
3A7
2J2
4A11
4B1
4F12
4F2
4F3
4F8
5A1
8A1
24
26A1
26B1
27B1
2A7
2R1
2S1
2U1
2W1
3A43
4A22
4F11
4F22
4V2
4X1
4Z1
20
26C1
27C1
a
This classification is somewhat arbitrary (e.g., P450s 1B1 and 27A1 could be grouped in two
different categories).
Source: Adapted from Guengerich (2003a, 2004).
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Basis of Regulation
In the 1960s and through the 1970s, the molecular basis was poorly understood,
largely because the technology was primitive by today’s standards. Some of today’s
students may not realize this, but sequence analysis for DNA was not possible until the
late 1970s (Maxam and Gilbert, 1977; Sanger et al., 1977). The concept of enzyme
induction in animals and even in human clinical situations was known since the 1950s
(Conney et al., 1956; Remmer, 1957), but until 1980 there was little solid evidence that
synthesis of new mRNA and protein occurred (Estabrook and Lindenlaub, 1979). With
the development of work in the steroid receptor field, a general view developed that
these models (i.e., ligand binding by a cytosolic receptor, transport to the nucleus,
interaction with nonhistone chromatin proteins to initiate or enhance transcription)
would explain P450 regulation.
Interestingly, the first extensively characterized regulatory system was the Ah locus
system (Nebert and Gelboin, 1968; Poland et al., 1976), which has similarities to the
steroid receptor model, but also many differences (different gene families, use of
heterodimers of different families, etc.) (Bradfield, 2004; Waxman, 1999). Since the
late 1990s, the analysis of the steroid receptor superfamily orphans has led to the
recognition of a number of these as being involved in the regulation of P450s [e.g.,
PXR, CAR, FXR, LXR, PPAR, and the retinoid-binding heterodimer partner RXR
(Bradfield, 2004; Kliewer et al., 1998; Nebert and Russell, 2002; Russell, 2000;
Waxman, 1999)]. In a sense, it seems almost ironic that the field came back to one in
which these steroid receptor proteins dominate.
Knowledge of the roles of these receptors and the AhR/ARNT system (Hankinson,
1995) has led to the use of simplified reporter systems to use in screening for P450
induction with new drugs, which has relevance to predictions of drug interactions and,
in some cases, tumorigenicity. However, our understanding of the basic components of
these systems becomes complicated with the details of coactivators and other accessory
systems. This information is becoming available, along with more knowledge about
roles of cytokines and other regulators (Delesque-Touchard et al., 2000). Further, the
P450s involved in steroid metabolism are regulated quite differently than those P450s
devoted to xenobiotic oxidation, as would be expected (Kagawa and Waterman, 1995).
Although more details remain to be elucidated in all systems and we will probably be
surprised by some regulatory systems, we certainly know much more than we did in the
early days of P450 research.
Catalytic Mechanism
Work in this area has never been easy. The chemistry of oxidation of rather
inactive molecules is not obvious and interested many organic chemists, not only with
P450s, but in general because of the practical applications for synthesis (Sheldon and
Kochi, 1981). Early literature in the field included proposals of mobile oxidants (e.g.,
superoxide anion being generated by P450 (or even NADPH-P450 reductase) and
reacting with a substrate fixed in a particular position (Aust et al., 1972; Strobel and
Coon, 1971). A key development in the field was the realization that an Fe –O complex
was the oxidizing species formed in the enzyme and then reacted with the bound
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substrate (Groves and McClusky, 1976), a process amenable to more control. The
findings of Groves and Coon (Groves et al., 1978) were seminal in this regard and still
dominate the general views of P450 chemistry, although some details are still under
consideration (Ortiz de Montellano, 1995, 2004).
The general mechanism can be described, at least in my own thinking, as an
‘‘odd-electron’’ process (Guengerich and Macdonald, 1984), involving the transfer of
one electron or hydrogen atom to generate an intermediate complex that collapses
by recombination, sometimes with accompanying rearrangements prior to or
following product formation:
RH þ FeO3þ ! R þFeOH3þ ! ROH þ Fe3þ
RH þ FeO3þ ! RHþ þ FeO2þ ! R þFeOH3þ ! ROH þ Fe3þ
This basic mechanism can be used to rationalize most of the known P450 oxidation
reactions (Guengerich, 2001a; Guengerich and Macdonald, 1984; Ortiz de Montellano,
1995). The regioselectivity and stereoselectivity of product formation are generally
agreed to be functions of chemical reactivity at individual sites and how the P450
orients the substrate (e.g., the juxtaposition in the active site), which are not necessarily
independent parameters. Multiple oxidants are another potential factor (vide infra).
With this knowledge, it has been possible to make some predictions, at least within
families of similar compounds.
Three-Dimensional Structures
I first learned of the existence of some crystals of bacterial P450 101 (in Gunsalus’s
laboratory) in 1973. The first structure of this protein was reported more than 10 years
later (Poulos et al., 1985). The availability of the first P450 101 structures answered
some important questions. The heme axial ligand was Cys357; this enzyme could only
bind the substrate by opening the entrance channel, and small but important structural
changes occurred during changes in redox states (Poulos et al., 1987; Raag and Poulos,
1989). The structure also provided valuable insight about amino acid candidates for sitespecific mutagenesis (e.g., Thr252, Asp251) (Imai et al., 1989; Martinis et al., 1989).
Since that time, at least 12 microbial P450 structures have been published, several
of these at very high resolution. More recently, some rabbit (Scott et al., 2003; Wester
et al., 2003a; Williams et al., 2000) and human (Schoch et al., 2004; Wester et al.,
2003c; Williams et al., 2003) structures have also appeared, with all the available
human structures coming from the 2C subfamily to date. These structures do not reveal
all the details of how P450s deal with their substrates, but collectively the structures
have told us much about what P450s are (Johnson, 2003). The overall folds are similar,
and from a distance the P450s do not look very different. In the case of P450 101, we
have a set of structures showing some structural changes as the enzyme progresses
through the catalytic cycle (Schlichting et al., 2000) and also structures of the enzyme
holding several different substrates (Chen et al., 2002; Raag and Poulos, 1991).
Although the work with the human P450s is relatively new, the work offers potential
for understanding the selectivity of a P450 for different drugs and also the regioselectivity
of oxidation (Aldridge, 2003; Wester et al., 2003a; Williams et al., 2003).
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Significance of P450s in In Vivo Metabolism
Indirect evidence for the significance of P450 alterations in metabolism goes back
to the 1950s (Miller et al., 1958; Richardson et al., 1952). The demonstration of
multiple forms of P450 beginning in the late 1960s led to further interest in the
relevance of the complexity of P450s. The severity of inborn errors of metabolism had
been known in the field of endocrinology for some time (Miller, 1986; Nebert and
Russell, 2002), and these reactions became recognized in terms of their P450
components (Chen et al., 1986; Chung et al., 1986). Some of the P450 deficiencies are
lethal (Nebert and Russell, 2002).
Another seminal effort was the work of Smith demonstrating the monogenic
control of the oxidation of certain drugs (Mahgoub et al., 1977). This effect, now
understood as the CYP 2D6 polymorphism, was critical in establishing that a loss of a
single P450 could be important because of its selectivity in the oxidation of a particular
drug. The in vivo system was characterized with the isolation of the P450 2D6 protein
(Distlerath et al., 1985; Gut et al., 1986), the cDNA (Gonzalez et al., 1988), and the
gene (Skoda et al., 1988). Polymorphisms have now been discovered with many more
of the ‘‘xenobiotic-substrate’’ P450s (Nagata and Yamazoe, 2002; http://www.imm.
ki.se/CYPalleles), and characterizing potential problems with induction and polymorphism is a major issue in the development phase with new drug candidates (Evans and
Relling, 1999; Guengerich, 2003).
The development of transgenic methods has provided an opportunity to demonstrate
the in vivo significance of individual P450s (Gonzalez and Kimura, 2003). Comparisons
of knockout mice with humans are not always particularly easy to interpret because of
gene redundancy and the lack of completely orthologous relationships among the
subfamily proteins (Nelson et al., 1996). Thus, developing mouse models for the human
xenobiotic-substrate P450s has often been difficult. However, it has been possible to
demonstrate strong modulation of toxicity and cancer in some knockout mouse models
(Buters et al., 1999; Gonzalez and Kimura, 2003).
Translation to Human Research
When I was deciding about research areas for further training near the end of my
graduate studies in 1972, I was attracted by P450s because I beleived that the field
might have potential relevance someday (in addition to being inherently interesting). In
the 1970s, research in the pharmaceutical industry consisted mainly of in vivo studies
of drugs with laboratory animals, and academic research in this field involved in vitro
studies with liver or other tissues (usually microsomes, occasionally purified enzymes)
isolated from laboratory animals (usually rats and rabbits). A key development was the
increased availability of human liver and other tissues for in vitro work (Distlerath and
Guengerich, 1987). The purification of the human P450s (Distlerath and Guengerich,
1987), and later the cloning and heterologous expression of the human P450s
(Gonzalez, 1989), has provided the ability to focus on human systems. Studies can be
done on the oxidation of drugs and on induction in cultured cells of human origin.
Today in vitro studies are done with human P450s in industrial and academic research,
and most work with animal P450s is done only to support specific studies (e.g.,
metabolism or toxicology) relevant to the use of those species, not with the view that it
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will necessarily be of direct application to human studies. The concern is not whether
in vitro studies with human P450s can be done, but exactly how relevant they will be to
in vivo situations.
Polymorphism and Effects
Some points were already made under the heading of ‘‘Effects on In Vivo
Metabolism’’ (vide supra). Polymorphisms have been shown to have dramatic (and
sometimes even lethal) effects on drug metabolism in humans (Ritchie et al., 1980).
After more information became available, polymorphisms were considered a serious
problem. We do generally recognize that polymorphisms in the drug-metabolizing
P450s can be a problem in certain cases, particularly if therapeutic windows are poor.
One might wonder why approximately one-third of the drugs marketed today are
oxidized by a highly polymorphic P450 (P450 2D6) (Evans and Relling, 1999;
Guengerich, 2003). The answer is that the process of developing the older drugs, if not
necessarily efficient, showed some self-selection in discriminating against those drugs
in which the polymorphism would lead to very serious side effects in many patients
(i.e., having poor therapeutic windows).
Today the general view is to avoid drug candidates that involve a major
involvement of a highly polymorphic P450, giving preference to candidates of similar
efficacy that have metabolism distributed among several P450s (Guengerich, 2003).
This appears to be a logical approach, and we expect the large fraction of drugs
metabolized by P450 2D6 (Evans and Relling, 1999; Guengerich, 2003) to decrease in
the future.
WHAT ISSUES HAVE NOT BEEN RESOLVED?
Posttranslational Modification
Relatively little research in this area has been done with P450s, with the exception
of phosphorylation in some cases. Glycosylation has not been found in the cases it has
been examined (Armstrong et al., 1983; Guengerich, 1978). The only report of
glycosylation involves P450 19A1 (the aromatase) (Shimozawa et al., 1993), except for
a small amount of sugars in early preparations of rabbit P450s (Haugen and Coon,
1976). Presumably, extensive posttranslational modification does not occur because
catalytically active P450s can be expressed in bacteria (Barnes, 1996; Guengerich et al.,
1996), including P450 19A1 (Chen et al., 2003; Kagawa et al., 2003). Nevertheless, the
possibility of posttranslational modification exists, if not in the bacteria then possibly in
mammalian cells. Phosphorylation has been mentioned and reasonable evidence has
been published for a role in the regulation of rat P450s 2B1 and 2E1 (Correia, 2003),
although the apparent effect of phosphorylation is not all or none. Another issue is that
limited effort has been devoted to analyzing P450s directly recovered from tissues.
New proteomics procedures are impressive, and the identification of modified
peptides of P450s should be a relatively straightforward procedure (Hansen et al.,
2001). A clear advantage of mass spectrometry is the ability to search for many
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different modifications simultaneously, in that more than 100 protein modifications
are known.
Ubiquination of some P450s has been reported (Korsmeyer et al., 1999), but
relatively few P450s have been examined in this regard. P450 degradation is important,
but the studies in this area are still quite limited (Kimzey et al., 2003).
One interesting area is the ‘‘partial’’ trafficing of microsomal P450s to unusual
organelle sites. Avadhani’s group first reported the presence of microsomal P450s in
liver mitochondria (Niranjan et al., 1984) and showed that they can work efficiently
with adrenodoxin as an electron donor, as well as with the flavoprotein NADPH-P450
reductase (Anandatheerthavarada et al., 1998). In recent collaborative work with my
group, Avadhani’s laboratory has been able to demonstrate this process with P450 2D6
in human liver (unpublished results). Further polymorphisms in the region near the
N-terminus affect the trafficking of P450 2D6 to the mitochondria.
Further Details of Regulation
The field of gene regulation seems to be never ending. With many of the P450s,
major control elements have now been identified, but it is not really valid to think of
these control elements as simple on/off switches. Regulatory systems generally involve
multiple elements (e.g., a receptor-binding site and a binding site for factors that impact
tissue specificity). Further, protein factors often interact with each other or with
additional proteins (e.g., coactivators). An example of the complexity of a P450 system
is seen with P450 3A4 and the roles of PXR and HNF-4 (Tirona et al., 2003).
Thus, we need to be thinking of regulatory pathways in the context of
multidimensional networks, not simply a single element or a linear pathway. Another
issue is that cell-based systems can be set up as reporters for assays of induction, but
these systems are not going to be perfect because of these complexity problems. Recall
that most permanent cell lines were derived from cancer cells, or cells that have
developed abnormalities. In principle, some of these problems may be overcome by
appropriate co-expression in some cases. We are also beginning to see some of the
complexity in P450 systems that involve endobiotic chemicals, where transformation of
ligands by one P450 can regulate the expression of another (Goodwin et al., 2003;
Staudinger et al., 2001). Still another problem is understanding the regulation of the
steady-state levels of the regulatory proteins that control levels of individual P450s.
Finally, the regulation of many P450s has not been addressed. This list includes
many of the human P450s; with some of these, the sites of expression have not been
reported (Table 1) (Guengerich, 2003). Another point is that we still have a limited
view of the regulation of P450s in plants and microorganisms, and may expect to find
considerable novelty in the pathways. For instance, the interactions between plant and
insect P450s provide an interesting picture of conflict, survival, and synergy in biology
(Schuler, 1996).
Component Interactions
This is an old area of research and yet many questions have not been answered.
We have a fairly good view of how the mitochondrial P450s interact from the work of
Kamin’s group (Lambeth et al., 1980). Of course, the components of the bacterial P450
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101 system have been extensively studied for over 30 years (Mueller et al., 1995;
Tyson et al., 1972). From these, we still find interesting new information with newer
methods (e.g., how putidaredoxin binding alters the structure of P450 101) (Tosha
et al., 2003).
Our understanding of the microsomal P450s is still far from perfect. In the 1960s,
the P450, NADPH-P450 reductase, and phospholipid components were identified (Lu
and Coon, 1968) and subsequently a role of b5 was implicated with some P450s
(Hildebrandt and Estabrook, 1971).
Our current model has the P450, NADPH-P450 reductase, and b5 moving about
and colliding through the phospholipid membrane, using the tails of each protein as
membrane anchors and using protein –protein interactions to make contacts and
facilitate electron transfer. We do know some things about the recognition sites for
some P450s and the reductase (Voznesensky and Schenkman, 1994). However, there is
still an element of mystery about the stoichiometry in the endoplasmic reticulum, which
is usually – 1 : 0.05 : 1 for total P450 : NADPH-P450 reductase : b5. Exactly how does
this system work in this apparent deficiency of reductase? We often study P450
reactions in systems with excess reductase to focus on the rates within the P450 system,
but several lines of evidence suggest that rates of binding and dissociation of the
components are not that fast, either in constituted systems (Guengerich and Johnson,
1997) or in microsomes, where biexponential reduction kinetics are observed. One
conclusion is that electron transfer to P450 (in microsomes) is not rate limiting for slow
reactions, but is for the faster ones. However, even slow reactions can show high
kinetic deuterium isotope effects (Guengerich et al., 1988).
What does b5 do? b5 can stimulate or inhibit P450 reactions (or have no effect)
(Gorsky and Coon, 1986). Pompon has offered an explanation involving distinctions
(individual P450 and substrate) in terms of the poise of redox potentials (Pompon and
Coon, 1984). Good evidence exists for a role of electron transfer (to FeO22+) in the
case of some P450s (Guengerich and Johnson, 1997; Pompon and Coon, 1984). An
unexpected finding in our own group was that apo-b5 (devoid of heme and incapable of
electron transfer) is as effective as (holo) b5 in stimulating the catalytic activities of
several P450s (Yamazaki et al., 1996a,b, 1997; Yamasaki et al., 2002). These findings
with P450 3A4 (Yamazaki et al., 1997) have been extended to P450s 2A6, 2B6, 2C8,
2C9, 2C19, 3A5, 4A7, and 17A1 (Auchus et al., 1998; Loughran et al., 2001;
Yamazaki et al., 1997, 2002). One problem in studying these systems is that the
(productive) binding of P450 and b5 is a very slow process (Guengerich and Johnson,
1997) and certain kinetic experiments are not really feasible. In microsomes, it is not
really possible to work with apo-b5, and kinetic analysis is complicated by the inability
to distinguish individual P450s. Thus, there is still ambiguity about what b5 really does.
Another issue involving component interactions is the competition of individual
P450s for reductase (Kaminsky and Guengerich, 1985) and the unexpected ability of
one P450 to stimulate the activity of another (Yamazaki et al., 1997). Backes has
presented some interesting results regarding such interaction of rabbit P450 1A2 and
2B4 (Cawley et al., 1995) and some possible explanations (Backes et al., 1998).
Work with microorganisms is beginning to reveal some unusual assays of
components (e.g., a single protein with a flavin and an iron-sulfur component) (Roberts
et al., 2003). Another example is Streptomyces coelicolor, with 18 P450s, 6 ferredoxins, and 3 (flavin) reductases (Lamb et al., 2002). Exactly how these compounds
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interact is a current interest in my own group, in collaboration with my colleague
Prof. Waterman.
Universality of P450 – Ligand Structures
X-ray crystal structures of P450s and P450– ligand complexes are exciting to see
and have provided insight into the interactions between P450s and their substrates. One
of the greatest hopes of this work is that it will be possible to predict the
regioselectivity of oxidation of other molecules.
P450 101 binds the preferred substrate camphor tightly, and the crystal structure of
the complex appears to be quite useful in rationalizing the regioselectivity of camphor
hydroxylation (Poulos, 1988). With the extensive structures of P450 102 available
(Peterson and Graham-Lorence, 1995), the structures still do not predict the correct
regioselectivity of hydroxylation (Capdevila et al., 1996).
Recently, intense interest has developed in the determination of X-ray crystal
structures of human P450s (Schoch et al., 2004; Wester et al., 2003; Williams et al.,
2003), following the successful determination of a rabbit P450 2C5 structure by
Johnson’s group (Williams et al., 2000). The driving force for commercial development
is the belief that establishment of a structure of a particular P450 with a bound ligand
will predict the mode of binding of other ligands to the same P450 (Aldridge, 2003).
That may or may not be the case. It is of interest to note that Johnson’s group has
identified two modes of binding of progesterone and also of a rationally developed
P450 2C substrate in P450 2C5 (Marques-Soares et al., 2003; Wester et al., 2003a).
[Another interesting wrinkle is the presence of ‘‘adventitious’’ palmitate bound in the
dimer interface of P450 2C8 (Schoch et al., 2004); the meaning for the microsomal
system is unclear.] Knowing the binding mode for the interaction of a substrate may or
may not accurately predict other configurations of the same molecule in the active site.
The issue will probably be more of a problem in situations with lower substrate
affinity. The problem is also discussed here in the context of the possible ‘‘escape’’ of
substrates from the active site late in the catalytic cycle, as evidenced by some of the
competitive kinetic deuterium isotope effects and ‘‘metabolic switching.’’
Do the crystal structures of P450 –ligand complexes predict structures for other
ligands? The question should probably be considered still open at this point (as well as
the real question, do the results predict hydroxylation sites?). The fits will probably be
tighter with closely related compounds and perhaps poorer with distantly related
compounds. Having raised caveats, the axiom is still that it is better to have structures
than no structures (or only modeled structures).
Details of FeO Chemistry
As indicated earlier, there is now general agreement that the oxidation reactions
proceed via high valent iron –oxygen complexes, as opposed to mobile oxygen species
that diffuse within and out of the enzyme. Most of the oxidation reactions can be
rationalized in the context of an intermediate first proposed by Groves et al. (1978) and
generally depicted as either FeV O, FeIV OP+ [where P = the heme porphyrin, a
valence distribution adopted from peroxidase chemistry (Dolphin and Felton, 1974)
that avoids the high charge on the iron and is probably a better representation of the
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true electronic state], or FeO3+ (a representation I prefer and avoids the specifics of
change localization).
The FeO3+ chemical mechanisms have generally served us well in rationalizing
P450 reactions (see Fig. 2). One set of reactions that does not fit well is the last of the
three steps in the (P450 19A1) aromatase process, which converts androgens to
estrogens. A proposal was made to account for this reaction with an iron hydroperoxy
intermediate (Akhtar et al., 1994; Cole and Robinson, 1991). Coon and Vaz proceeded
with a series of experiments that established that other P450s, the typical xenobioticsubstrate type, could also catalyze similar oxidative deformylation reactions with
substrates (Vaz et al., 1994).
Rearrangement reactions with strained alkanes have been long used as diagnostic
reagents for radical reactions in P450s (Hanzlik and Tullman, 1982; Hanzlik et al.,
1979; Macdonald et al., 1982; Stearns and Ortiz de Montellano, 1985). Efforts to use
such systems to ‘‘clock’’ rates of oxygen rebound (to incipient radicals) provided some
realistic constants (Ortiz de Montellano and Stearns, 1987), but studies with ‘‘faster’’
clocks by Newcomb led to the conclusion that the lifetime of a putative radical would
have to be so short so as not to consider it a true intermediate (i.e., low fraction of
rearranged product) (Newcomb et al., 1995). Newcomb has interpreted these and some
other results as evidence that P450s act, at least in part, by a mechanism that involves a
concerted (as opposed to stepwise) mechanism and the involvement of FeOOH or
FeO2 instead of FeO3+ (Newcomb et al., 2003; Toy et al., 1998). Others have not
accepted this view and provided further support for a central role of the FeO3+ entity in
catalysis (Auclair et al., 2002). Shaik’s group has used theoretical approaches
Figure 2.
Generalized catalytic cycle for P450 reactions.
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and presented a two-state theory, in which FeOO and FeOOH are generally not
oxidants but everything proceeds from FeO3+, with a dual spin-state system that
generates products characteristic of either a concerted or a stepwise mechanism (Shaik
et al., 2002).
Definite answers do not come easily in this field because of the intractability of
high-energy intermediates such as FeO3+ (and the other species). Some of the
approaches have caveats so lack of rearrangement of a substrate may be dictated by
binding forces within the enzyme that are not seen in solution (Frey, 1997). Certain
P450 Thr/Ala mutants have been used with the pretext of changing the protonation
state of the FeO2 complex, but we cannot be completely sure the mutation has not
introduced other defects into the enzymes (Yun et al., 2000). The two-state FeO model
presented by Shaik (Harris et al., 2000) has a certain intellectual attraction in
explaining several phenomena, one of which might be the varying kinetic hydrogen
isotope effects we have seen within the same substrate (and enzyme) (Yun et al., 2000,
2001). Unfortunately, there are few if any means of addressing this proposal with
experimental studies (as opposed to theoretical). Thus, we may be in a situation in
which theoretical arguments may be dominant for some time.
Why Do Different P450s Vary in Reaction Rates?
This is only part of the question; it can be expanded to consider why a single P450
catalyzes distinct reactions of a single substrate at different rates. A simplistic answer
might be that the position of a target atom is closer to the FeO center, but is this the
only answer? We have rates of P450 reaction that vary from 104 min1 (P450 102
hydroxylation of arachidonic acid) (Boddupalli et al., 1990) to many reports of less
than 1 min1. Indeed, I have reviewed papers with reports of these 1 min1 rates that
claim to have identified a physiological role of a P450 in a tissue (they may be correct,
even if slow). To some extent, we (or, more appropriately, the pharmaceutical industry)
may be screening against rapidly oxidized P450 substrates in the drug development
process. Further, the high levels of many P450s present in the body would be a
problem if very high rates of oxidation were the norm for sterols, vitamins, and other
physiological substrates.
Let us return to the question at hand: what really determines the rate of a P450
reaction? We consider the general catalytic scheme of Fig. 2; the time-honored tradition
in enzymology is to consider which steps are rate limiting. I will not discuss the issue
at length here. With the ‘‘fast’’ P450 101, the input of the second electron (from
putidaredoxin) is generally considered to be rate limiting in camphor hydroxylation
(Mueller et al., 1995). However, with ‘‘slow’’ substrates for the same enzyme, one can
now see high intermolecular noncompetitive deuterium effects, as might be expected
(Kadkhodayan et al., 1995). In the fast P450 102 hydroxylations, the rate-limiting step
has not been identified, but may well be an electron transfer (the complexity of the
unimolecular electron transfer process complicates the analysis, particular when the
rates move into the range of electron transfer between the flavins).
What about ‘‘slow’’ P450s and ‘‘slow’’ or ‘‘moderately slow’’ substrates? We
have provided some of our own thoughts in the primary literature (Bell and
Guengerich, 1997; Bell-Parikh and Guengerich, 1999; Guengerich and Johnson, 1997;
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Guengerich et al., 2002; Yun et al., 2000) and reviews (Guengerich, 2001a, 2002b). To
date, we have several scenarios. In the oxidation of ethanol and acetaldehyde by P450
2E1, a slow step occurs after product formation (required to explain the burst kinetics),
but is not product release per se (Bell and Guengerich, 1997; Bell-Parikh and
Guengerich, 1999). Contrary to what is often assumed in the literature, the rate of
electron transfer from the NADPH-P450 reductase to the P450s does not seem to be
rate limiting in most cases (Guengerich and Johnson, 1997) (in microsomes, the rate
might be limiting with some of the faster substrates due to the substoichiometric
concentration of reductase, although evidence is rather limited).
Kinetic hydrogen isotope effects provide a means of obtaining information about
the rate-limiting nature of the C –H bond-breaking step (Fersht, 1999; Walsh, 1979).
The most directly relevant type of study in this regard is a noncompetitive
intermolecular experiment (Fig. 3) (Northrop, 1982). We have measured such isotope
effects of 2 – 4 for P450s 1A2 and 2D6 (Guengerich et al., 2002; Yun et al., 2000,
2001). These results help establish C –H bond breaking as a partially rate-limiting step.
Kinetic simulations have been done on both of these enzymes and fit with the concept
that C – H bond breaking is partially rate limiting (Guengerich, 2001a, 2002b).
Sometimes the literature appears to imply that P450 3A4 is a rather open ‘‘hole’’
that substrates enter and rattle around in, with the regioselectivity of hydroxylation
dominated by the chemical ease of hydrogen abstraction at individual atoms (Smith and
Jones, 1992). If this view of P450 3A4 is really valid, then we might expect a relatively rapid reaction catalyzed by the enzyme (e.g., testosterone 6b-hydroxylation)
(Guengerich et al., 1986) to show a high kinetic deuterium isotope effect. We have
done such experiments and find relatively low isotope effects (2 –3) (Krauser and
Guengerich, 2003). In the work, we find a higher isotope effect (7) for a slower
reaction catalyzed by the same enzyme, 2b-hydroxylation. The results are reminiscent
of another case, where human P450 1A2 shows a low isotope effect (2 –3) in the
O-dealkylation of 4-alkyoxy acylanilides and high isotope effects (14) for acetol
formation (Guengerich, 2001a; Yun et al., 2000). One could simply say that the isotope
effect reflects the ease of forming the major product vs. minor ones, although the
difference in rates of production of the products is only four-fold (Guengerich, 2001a;
Yun et al., 2000).
Another issue is what steady-state kinetic parameters really mean in P450
reactions. From the previous discussion, we do not have a definite idea of what kcat
(Vmax) really represents in most cases. The identity of Km is much worse. Unfortunately,
the literature often equates this parameter with the Kd for substrate, and I have even
seen papers where only Km values are presented, as a measure of P450 preference for
substrates. The complexity of the Km term is revealed in several reactions catalyzed
by P450 2E1, where deuterium substitution produces an increase in Km but not kcat
(Mico et al., 1985). An explanation is provided with knowledge that burst kinetics are
operative: kcat is dominated by a step following product formation, and Km contains
the term of the rate of C – H bond breaking (Bell and Guengerich, 1997; Bell-Parik
and Guengerich, 1999). Closer inspection indicates that the parameter Km is also a
function of kcat, so Km is not an independent variable (Bell and Guengerich, 1997;
Guengerich et al., 1995). See Northrop (1998) for a good discussion about the
meaning of these parameters, which is usually not taught very well in biochemistry
classes today.
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Figure 3. Some kinetic hydrogen (deuterium) isotope effect experiments relevant to P450
research. (See also Cleland et al., 1977; Gillette, 1991; Gillette et al., 1994; Northrop, 1975, 1982).
A, Noncompetitive intermolecular experiment, yielding the isotope effect noncompk, which is a good
measure of the extent to which the C – H bond-breaking step is rate limiting in the overall reaction.
B, A competitive intramoleular experiment, yielding compk, a measure of the commitment
to catalysis and possibly the ability of the substrate to tumble within the active site. This is
best used when the two sites are equivalent (e.g., an N, N-dimethyl amine). C, Intramoleular
competitive experiment, in which Dk approximates the intrinsic isotope effect. [An alternative
method of estimating the intrinisic isotope effect involves the use of tritium and Northrop’s
equation (Northrop, 1982).] D, An intermolecular competitive experiment. As seen here, the effect
of the C – D bond is not sensed until the chemical reaction step involving the substrate. compk is an
estimate of the apparent isotope effect, but might have to be corrected for step e. At present, it is
not clear whether a free FeO3 + entity results or step e is involved. If the reaction shows total
commitment to catalysis (Northrop, 1982), then compk = 1. In principle, the total flux of the system
(comparing the rate of alcohol formation in part D with the ‘‘C – H only’’ component of part A)
should address this question. This scheme is intended only as a rough introduction and guide. As
indicated in the text, the systems are more complex and several questions remain to be answered
with P450s.
This discussion could go on, but suffice it to say that we still have a long way to
go in understanding P450 catalysis from the standpoint of why reaction rates differ, and
the answers will require more than static structures. A final point to make is that the
rates of nonproductive oxygen reduction are very important. These rates are slow in
comparison with the fast reactions catalyzed by some of the bacterial P450s, but faster
than rates of oxidation of the ‘‘slow’’ substrates by microsomal P450s. Kinetic
simulations show that both kcat and Km (for an oxidation reaction) can be influenced by
rates of these nonproductive reactions (Guengerich, 2001a).
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Details of Reaction Steps
The catalytic cycle shown in Fig. 2 is a minimal mechanism, although it already
looks rather complicated to students. As mentioned earlier, most of the steps probably
also involve conformational changes, which we have some information about
(Schlichting et al., 2000). Whether these structural changes are necessary for the
chemical steps (or how tightly they are associated) is not very clear yet.
Recently, I have given some thought to several of the individual steps in the
catalytic cycle, although not necessarily working on all of them. I would like to
‘‘revisit’’ some of these and the earlier literature about them, with the hope of
stimulating interest that will lead to a better understanding.
Step 1 (Fig. 2) is usually considered rather straightforward, but is it? Substrate
‘‘on-rates’’ are generally taken to be diffusion-limited second-order reactions with rates
107 –109 M1 sec1 (Fersht, 1999; Johnson, 1992). These are sufficiently fast to
preclude analysis without stopped-flow or jump techniques, so not many people study
these. However, there is considerable speculation about multiple ligands in P450
binding sites, particularly with some of the drug-metabolizing P450s such as P450 3A4
(Harlow and Halpert, 1998; Hosea et al., 2000; Shou et al., 2001). If this is the case, is
substrate binding really a simple process, or can we begin to see details with the
addition of a second ligand? Perhaps another look is in order.
We presented work on the reduction (step 2) of several human P450s several years
ago (Guengerich and Johnson, 1997). I am not sure all aspects of the process have been
solved. In reconstituted systems, some P450s show monoexponential kinetics and some
show biexponential kinetics (Guengerich and Johnson, 1997). The difference is
probably due to rapid reduction of only the P450 population in the binary complex
(with reductase), although independent proof of this is admittedly lacking.
The technical difficulties of studying step 4 (Fig. 2) are a problem, and any studies
of this process are usually rather indirect. Unfortunately, we cannot simply mix P450
(FeO22+) and reductase; studies on step 2 show that formation of a productive complex
is slow (Backes and Eyer, 1989; Guengerich and Johnson, 1997).
Steps 5 to 8 (Fig. 2) are even harder to study, although these are the ones we want
to know more about. There are some options to studying steps with dyes and flash
photolysis (Dunn et al., 2001), but still one has to consider any results in the context of
consideration about the effects of binding to auxiliary proteins, (i.e., the redox partners)
(Tosha et al., 2003).
Step 9 is the parallel to step 1 (Fig. 2). I have already discussed some of our results
with P450 2E1 and the burst kinetics that implicate a rate-limiting step after product
formation (Bell and Guengerich, 1997; Bell-Parikh and Guengerich, 1999). We have
not observed this behavior with P450 1A2 or 2D6 (Guengerich et al., 2002; Yun et al.,
2000), and we have not examined other P450s yet. The slow step is not product release
per se, because the products (acetaldehyde and acetic acid) do not have particularly
high affinity for P450 (Bell-Parikh and Guengerich, 1999). We hypothesize that the
slow step is a conformational change not directly associated with product release, but
more work is needed to characterize this process.
The previous discussions bring up a point that developed during considerations of
results of competitive kinetic deuterium isotope effect studies (Fig. 3). The competitive
intermolecular isotope effects are generally high (i.e., the intrinsic or noncompetitive
isotope effects are not strongly attenuated). To put these results into the more classic
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descriptions of isotope effects (Northrop, 1975), we have low commitment to catalysis.
The FeO complex may be committed to proceeding through some event, but it could be
formation of reduced oxygen (e.g., H2O formation). The presence of a significant
deuterium isotope effect and ‘‘metabolic switching’’ to yield enhanced formation of
alternate products can only be rationalized by a release of substrate and rebinding in a
position more favorable for oxidation. Metabolic switching alone could be rationalized
by tumbling, but the intermolecular competition experiments argue that the substrate
leaves the enzyme and then returns (Fig. 3). For example, the intermolecular
competitive isotope effect for methacetin by P450 1A2 was 3.1 (cf. an estimated
intrinsic isotope effect of 5.4) (Yun et al., 2001).
This relatively simple experiment, which is certainly not unique in the literature
(Northrop, 1982), has fairly profound implications and relates to some of the discussion
of the universality of P450– ligand structures. We generally think of step 1 as the
discrete substrate binding step and step 9 as the product binding (release) step (Fig. 2).
However, could the exchange happen at the FeO3+ step? Let’s consider this, even if
it might seem intuitively unrealistic. Give the complex the same Kd that we have for
the ground state P450 (e.g., 10 5 M). Using a typical diffusion-limited on-rate for
substrate of 108 M1 sec1 (Fersht, 1999; Johnson, 1992), we have an off-rate of
103 sec1. This is still much faster than any known P450 reaction, and remember that
the existence of any hydrogen isotope effect argues that C –H bond breaking is not
going to be incredibly faster than the overall kcat. So we might have plenty of time to
release the substrate. A corollary is that, if we are operating at a substrate concentration
of 10 mM (Km), the on-rate is also 103 sec1. Precedent for a difference in the affinity
in redox forms of a P450 for a ligand is seen in our work with P450 3A4; the ferrous
form had 10-fold less affinity for a peptide inhibitor than did the ferric form (Hosea et
al., 2000). Expanding the argument, we can begin to consider the dissociation
equilibrium at most other steps in the catalytic cycle of Fig. 2. Now the system really
gets complex.
This scenario is predicated by the possibility that we might really have a more
‘‘gated’’ system in which the enzyme ‘‘locks’’ the substrate and keeps it held more
tightly through the reaction steps. This is in effect an ‘‘induced fit’’ model (Koshland
et al., 1966; Wester et al., 2003b). The conformational changes would have to be slow
to keep the substrate in. The competitive isotope effect results could be explained by a
simple ‘‘fizzling’’ of the activated complex (FeO3+ RD3 ! Fe3++ RCD3 in Fig. 3D),
rather than an exchange of ligands and an unloaded FeO3+ complex.
At this time, we do not have enough data to draw strong conclusions about which
of the previous views is more generally sound. The metabolic switching indicates that
P450s do certainly have time to twist within the active site.
Finally, another point to be made is that the literature contains many postulates
about access channels, arising from either the crystal structures (Podust et al., 2001),
modeling (Winn et al., 2002), or mutagenesis (Scott et al., 2002). It would be
interesting to examine the kinetics of binding and release of substrates and products
with mutants at the appropriate sites.
Relevance to Cancer
One of the driving forces in the development of the P450 field has been the
relevance to cancer, particularly in the area of chemical carcinogenesis (Conney, 1982;
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Gelboin 1967). I have worked on the oxidation of chemical carcinogens myself and the
National Cancer Institute has generously financed much of my research effort. What do
we really know about the involvement of P450s in the development of human cancer?
We have excellent evidence that modulation of P450s can influence the risk of
experimental animals to cancer (Guengerich, 1988; Nebert, 1989). People vary
considerably in their complement of individual human P450s (Guengerich, 2003, 2004)
and, as mentioned earlier, these differences have clearly been shown to have the
potential for dramatic effects in drug toxicity (Smith, 1986) and in sterol-related
diseases (Nebert and Russell, 2002). In vitro experiments have clearly demonstrated
roles of individual human P450s in the activation of specific chemical carcinogens
(Guengerich and Shimada, 1991). Given all this, we might expect to be able to readily
demonstrate clear differences in cancer susceptibility due to P450 polymorphisms.
However, much of the evidence to support such relationships is either irreproducible
or simply weak (d’Errico et al., 1996). I have covered some of the problems with the
epidemiology elsewhere (Guengerich, 2003). Briefly, we are dealing with some nebulous exposure data and generally small numbers, at least relevant to typical clinical
drug trials. In may perspective, the evidence for a role in cancer should be considered
rather weak for P450s 1A1, 2D6, 2E1, 3A4, and 17A1 (Guengerich, 2003).
However, there are some relationships with more potential, at least in my opinion.
These include P450 1A2 and colon cancer, which may involve heterocyclic amines in
cooked meat (Lang et al., 1994). Another prospect, at least in Japan (where a gene
deletion is found in a sizeable segment of the population) is P450 2A6, which can
influence the amount of smoking (due to nicotine metabolism) and carcinogen
metabolism (due to nitrosamine activation) (Ariyoshi et al., 2002). Another prospect is
P450 1B1. This enzyme activates many classes of carcinogens, including polycyclic
hydrocarbons, heterocyclic amines, and estrogens (Shimada et al., 1996). Recently,
Kamataki’s group provided evidence that the long-studied trimodal inducibility of aryl
hydrocarbon hydroxylation activity in human peripheral blood cells, with some link to
cigarette smoking-induced lung cancer (Kellerman et al., 1973), actually reflects P450
1B1 and not P450 1A1 (Toide et al., 2003).
COOPERATIVITY
Two aspects of cooperativity are observed with P450s: 1) heterotropic
cooperativity is the (direct) enhancement of a catalytic activity by a molecule other
than the substrate; 2) homotropic cooperativity is the nonhyperbolic behavior (in
plots of v vs. S) seen with a single substrate. The observation of the heterotropic
phenomena is not new (Anders, 1971; Buening et al., 1978; Cinti, 1978). Since the
1990s, interest has developed with reports of both types of behavior with P450 3A4
and some other P450s. I will not add too much here because I spent a fair amount
of space covering this topic under the P450 3A4 heading in another chapter
(Guengerich, 2004). In addition to P450 3A4, cooperativity has been reported with
P450s 1A2 and 2C9, and some other P450 enzymes (Hutzler and Tracy, 2002; Miller
and Guengerich, 2001).
Having worked in this area for some time (Hosea et al., 2000; Ueng et al., 1997), I
do have concerns about the reliability of some of the reports in the literature based on
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results I have seen firsthand (and not published). If one uses the equation v = VmaxS/
S50 + Sn, the n values are generally low (1.5) in this field and attention must be given
to the number of points collected, particularly at the low substrate concentrations
(where the error is worst). Also, a common occurence is depleting the substrate concentration with too much enzyme and has the effect of artificially inducing sigmoidicity.
Several investigators, including my own lab (Hosea et al., 2000) developed models
with multiple (2 or 3) substrates occupying space in a large substrate binding site
(Domanski et al., 2001; Egnell et al., 2003). Although many equations are used
regarding substrate binding (Galetin et al., 2002; Shou et al., 2001), only a few
investigators have actually examined substrate binding (Harlow and Halpert, 1998;
Hosea et al., 2000; Miller and Guengerich, 2001), and even then stoichiometry has not
been evaluated. Most of the studies cannot rule out many of the more classic allosteric
models [e.g., see Segel, (1975)].
My personal opinion is that this field would benefit from more evidence for
biological relevance and from application of physical approaches, neither of which are
easy. Evidence for homotropic cooperativity has been obtained with cultured
hepatocytes (Witherow and Houston, 1999), but showing this phenomenon in vivo
would be difficult. Heterotropic cooperativity has been demonstrated with diclofenac
and quinidine in monkeys (Ngui et al., 2000). Little has been published regarding
physical studies with the exception of an interesting P450 3A4 study with pyrene by
Atkins (Dabrowski et al., 2002), which provides good evidence for the existence of
interacting pyrene molecules bound to the enzyme. The Astex company has reported
that a crystal structure of P450 3A4 has been obtained (Aldridge, 2003); however, they
have not provided the actual structure or indicated what ligands have been used. The
Astex structure of the P450 2C9 warfarin complex showed only a single warfarin
molecule, but no real evidence for cooperativity of warfarin hydroxylation has really
been presented (for P450 2C9).
EMERGING AREAS OF P450 RESEARCH
Newer Methods of Analysis of Ligand Interactions
One of the exciting aspects of X-ray crystallography is the ability to visualize the
docking of a ligand in a P450. However, as mentioned earlier, these snapshots do not
necessarily provide a complete picture. We have already seen that a P450 can bind a
single substrate in multiple ways (Wester et al., 2003a; Williams et al., 2003), which is
not really surprising in light of known results about multiple products. Ideally, we want
to characterize dynamic interactions in an experimental mode.
Some relatively new techniques offer potential in achieving the goal. Complete
nuclear magnetic resonace (NMR) analysis of a three-dimensional structure of a P450 is
still probably outside the realm of possibility, at least for the aggregating microsomal
P450s. However, some new NMR techniques have been developed that would allow
calculation of structures of P450– ligand complexes by NMR by comparison with an Xray diffraction-determined structure of a single P450 –ligand complex. One technique,
using residual dipolar coupling, requires that all the NMR 1H resonances be established for
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the amino acids involved in liganding (Bax, 2003; Evenas et al., 2001). Once these are
established with a single ligand, alternate ligands could be mixed with the same P450
and the structures of the complexes might be calculated. Thus, one would not have to
determine the structure of every ligand – P450 complex by crystallography. The hurdles
to this process are 1) determining the crystal structure of a P450– ligand complex, and
2) establishing the NMR shifts of the amino acid residues involved in the docking.
Another approach that has apparently not been used yet is isothermal calorimetry
(Bradrick et al., 1996; Wiseman et al., 1989). This technique is relatively
straightforward and allows the analysis of protein-ligand interactions that are invisible
by other procedures. The method has the potential for quantitation of ligand binding,
which has been a deficiency in the work on cooperativity (vide infra).
Fluorescence methods have found only limited use to date. One of the interesting
applications has been the analysis of eximer starching in the binding of pyrene by P450
3A4 (Dabrowski et al., 2002). Fluorescence methods should, in principle, find great
utility in the analysis of multiple substrates within a single binding site, if indeed this
does prove to be the basis of cooperativity (Shou et al., 1994). Mention should also be
made of the potential of some of the elegant tether systems developed by Gray and his
associates (Dunn et al., 2001).
Roles of Conformational Changes in P450s
Within the Catalytic Cycle
We generally depict the P450 catalytic cycle in a form such as that shown in Fig. 2.
This is, as already mentioned, a minimal scheme and shows only the steps involving
chemical redox changes. However, many enzymes are considered to change
conformations in the course of their catalytic cycles (Fersht, 1999), and it seems
perfectly reasonable that P450s should. Indeed, some circular dichroism changes were
detected early in the field of P450 research (Yong et al., 1970).
In an elegant study with P450 101, Schlichting et al. (2000) were able to follow the
protein through the catalytic cycle and record X-ray structures at several steps. The
work demonstrates the subtle but real changes that occur in the active site residues at
every stage of the cycle. Thus, a more correct version of the scheme in Fig. 2 would
include these changes.
Other studies have not been so elaborate to date, but studies of some intermediates
have been done and show substantial changes of the P450s in binding substrates and in
moving from the oxidized P450 to the reduced (Li and Poulos, 1996; Poulos et al.,
1995). Moreover, in some cases, conformational changes are necessary just to open the
entry channel of a P450 to allow substrates to enter (Lüdemann et al., 2000). These
changes complicate our interpretation of the static structures recorded of (ferric) P450–
ligand complexes. For instance, the structure of the P450 102 – fatty acid complex
predicts the wrong stereochemistry for hydroxylation (Capdevila et al., 1996).
The real-time approach of Schlichting et al. (2000) may not be suitable for many
P450s, but it may be possible to obtain more X-ray structures of Fe3+ substrate, Fe2+
substrate, and Fe2+ substrate CO (analog of O2) complexes.
Another approach that has apparently not been applied to P450s is 1H/D exchange,
which can be done by mass spectrometry of peptides. This approach can be used to
map the relative motion at different parts of an enzyme.
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Functions of Orphan P450s
I personally find the issue of identifying function one of the major challenges in
the field and have some thoughts about how to proceed. We are now living in a world
of what I call ‘‘reverse biochemistry,’’ that is, biochemistry working in an order
opposite that I learned (Fig. 4). Thus, we can find genes rather quickly and, with
somewhat more effort, convert these into proteins via heterologous expression.
Unfortunately, we often have a more difficult time deciding what these proteins/
enzymes do, and comparing protein sequences and even folds is not always very
instructive. In the case of P450s, several have even been crystallized without any
insight into biological function. With many of the mammalian P450s (about 15 of the
human P450s) devoted to the oxidation of xenobiotics (Table 1), a function with
endogenous substrates can be ignored. However, in the microbial world, we are rapidly
characterizing and even crystallizing P450s faster than we can identify their functions.
This is also the case with the myriad of plant P450s (the Arabidopsis thaliana genome
has nearly 300 P450 genes). Even in the superfamily of the 57 human P450 genes
(Nelson, 2003), at least 15 of these have no known function at present (Table 1)
(Guengerich, 2003). I term these P450s orphans after the term used in the steroid
receptor superfamily, even though we have a paradox in that some of the xenobiotic
substrate P450s may have a function in protection of the organism under
certain circumstances.
How does one identify functions for these enzymes? In some cases, one can gain
biological insight through transgenic analysis (mainly knocking out genes and searching
for changes in function). Frankly, life is too short to search all possible substrates/
reactions one at a time. My own view is that we will need to devise strategies to search
in complex mixtures to identify reactions, with only limited bias as to what the
substrates and products are. Space does not permit a complete presentation of
strategies, but there are now very powerful mass spectrometry and computational
approaches available.
Figure 4.
Paradigms of traditional and contemporary biochemistry.
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Another aspect of the problem is that we do not know the redox partners a priori
in some of these systems (e.g., S. coelicolor with its 18 P450s, 6 ferredoxins, and
3 flavoprotein reductases) (Lamb et al., 2002).
Molecular Breeding
This may not be a familiar item yet, but I note that a journal with the name has
now appeared. The approaches of random mutagenesis and directed evolution may be
involved, but the overall goal of the plan is to breed better enzymes in that same way
that we would breed better plants or animals in agriculture. Work is already under way
in this area, some being more pedantic (our own) and some more practical.
First, some substantial improvements in P450 enzymes have been achieved using
rational design. These efforts have been largely restricted to some of the more
established bacterial models, for which many crystal structures are already available.
Wong’s group has been able to achieve some rather impressive improvements in P450
101, with regard to high catalytic activities toward ‘‘unnatural’’ substrates (Bell et al.,
2003; Chen et al., 2002). Arnold’s group has used P450 102 as a platform for the
development of catalysts that can be used to hydroxylate alkanes (Glieder et al., 2002);
this system has the advantages of not requiring auxiliary proteins and also having high
levels of expression. My own group has worked with human P450 1A2, using several
strategies, but particularly one based on the ability of new variants to bioactivate a
promutagen faster (Parikh et al., 1999). We have found achieving 10-fold improvements in catalytic efficiency relatively straightforward, but believe the approach has
still more potential; P450 reduction is still not rate limiting (Kim and Guengerich,
2004; Yun et al., 2000, 2001). An interesting aspect of our own efforts is that one can
achieve selective improvement of activity toward one substrate, but not a very closely
related one (Kim and Guengerich, 2004).
Utilization of P450s in Chemical Synthesis
I reviewed this area two years ago (Guengerich, 2002a), and the reader is referred
to that article for more information. P450s have been used in industrial processes for
more than 50 years, with the classic example of the 11b-hydroxylation of deoxycortisol
(Lednicer, 1998). This reaction, and many of the oxidations involving P450s in
industry, is done with a cultured microorganism and the specific P450 has not been
identified. An exception to this general rule is the P450 hydroxylase used to convert
compactin to the hypercholesterolemic drug pravastatin (Serizawa et al., 1983), for
which a crystal structure has been determined but not published.
I am not aware that there has been a dramatic increase in the use of P450s in
industrial research in the past two years (outside of the human P450 enzymes and drug
development), but I am confident that this field will grow. Some potential applications
in the synthesis of fine chemicals were presented earlier (Guengerich, 2002a). My own
group, collaborating with that of Laurent Meijer, has been able to use a human P450
2A6 mutant to synthesize sets of indigoid protein kinase inhibitors (indirubin
derivatives) with an order of magnitude lower IC50 values than indirubin itself
(Guengerich et al., 2004). Although the molecules resulting from these exercises should
still not be considered serious drug candidates, the work does prove that the concept
has potential.
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We are interested in applying this approach to synthesis and screening of new
antibiotics (Guengerich, 2002a). The general area has attracted interest, although much
of this has come from the area of polyketide synthases, not P450s. Ultimately, the
usefulness of new microbial P450s and polypeptide synthases may be combined to
facilitate antibiotic discovery. It is of interest to note the recent determination of crystal
structures for two bacterial P450s involved in vancomycin synthesis (Fedorov et al.,
2003; Zerbe et al., 2002).
CONCLUSION
I have spent more time and used more space than I originally intended to, but I
have still not covered every aspect of P450 research. I refer readers to the new edition
of Cytochrome P450, edited by Prof. Paul R. Ortiz de Montellano (to be published in
2004). Even after all these years, this is still a very interesting field and will continue to
be one. I have emphasized some basic research problems, but much also remains to be
done in the application of P450 research in practical problems in the pharmaceutical
industry and elsewhere. For instance, there are new mass spectrometry developments
that may permit very high-throughput analysis of P450 reactions with drugs (Benetton
et al., 2003; van Breemen et al., 1998).
When I started my own lab, I had concerns about entering what I believed was a
large and crowded field. I would encourage young scientists not to be overly concerned
about competition; there are plenty of resources and opportunities for good work in
some of the areas I outlined and in many others. At times, I have entertained the idea
of totally moving totally into other areas—I do work in other fields with DNA
polymerases, reactions related to toxicity, and other things I find interesting
(Guengerich, 2001b). However, I am now in charge of overseeing the biennial
international P450 meetings, so I really need to stay in the field. In all seriousness, I
still find the field full of research opportunities and will probably never leave the P450
business. I hope that my further contributions will be useful.
ACKNOWLEDGMENTS
This laboratory has been supported in part by U.S. Public Health Service grants
R01 ES01590, R01 ES02205, R01 CA33907, R35 CA44353, R01 ES10375, R01
ES10546, R01 CA90426, and P30 ES00267 over the past 29 years. I thank the ISSX
again for this award and the Xenotech company for its sponsorship. Of course, I thank
the many individuals who have worked in my group and wish them the best of success
in their own careers. I also thank my assistant K. Trisler for her help in this preparation
of this manuscript.
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