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J. Biol. Chem., Vol. 277, Issue 32, 28351-28363, August 9, 2002
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From the Department of Biological Chemistry, Medical School,
University of Michigan, Ann Arbor, Michigan 48109
Those unfamiliar with basic research in biochemistry and
related fields may assume that important discoveries are the result of
brilliant ideas that are single mindedly pursued until, many years
later, the answer is obtained, perhaps along with important biomedical
applications. The progress of science is almost always more haphazard,
as ambitious young scientists are influenced by their teachers, by the
cooperative or competitive work of others, the availability of new
techniques, and chance findings that may lead to different goals. Sixty
years ago as an undergraduate at the University of Colorado, I took my
first biochemistry course in the Chemistry Department taught by
Professor Reuben Gustavson and had the good fortune to be invited by
him to join his small research group studying steroid hormones. I had a
tremendous amount to learn but was fascinated from then on with
research and the possibility of making new discoveries.
In these reflections the influence of my mentors/teachers, whom I much
admired for their personal qualities and achievements, is acknowledged.
Although my research over the years has taken many unexpected turns, a
common thread has been an interest in biological oxidations,
particularly those not readily explainable according to the predictions
of organic chemistry. This curiosity has led to fundamental studies on
the properties and mechanism of action of cytochrome P450, now often
described as the most versatile biological catalyst known. Although
this was not my original goal, the mammalian isoforms of this enzyme
have turned out to be of biomedical importance because of their central
involvement in the metabolism of steroids, drugs, and chemical carcinogens.
William Cumming Rose was a dedicated and inspiring teacher and
an outstanding pioneer in biochemistry and nutritional science who
spent most of his career at the University of Illinois (1). Young Will
attended schools in small communities in North Carolina and South
Carolina until the age of 14, when the inadequacy of the education
caused his father to remove him from school and tutor him at home. He
had been introduced to Latin, Greek, and Hebrew and was well prepared
by the time he entered college. Will wished to attend a large
University, but his father thought his son at age 16 was too young and
convinced him to attend Davidson College in North Carolina, a school
for which he developed a lifelong affection. While in graduate school
at Yale University, Rose decided on the branch of chemistry he would
pursue, which was biochemistry, under the guidance of Lafayette Mendel
in the Sheffield Scientific School. In 1911, upon completion of his
Ph.D. thesis, Rose left Yale for an instructorship in physiological
chemistry at the University of Pennsylvania, followed by advanced study
with Franz Knoop at the University of Freiburg and then a faculty
position at the University of Texas in Galveston before he became
professor and head of the Division of Biochemistry in the Chemistry
Department at the University of Illinois. This provided a permanent and
very supportive home for his scientific career for the next 35 years.
In research Rose displayed a gift for meticulous experimentation and
for thoroughness and clarity in his publications. As a teacher he
imbued students who attended his carefully prepared lectures with
enthusiasm for biochemistry. The subject came alive with his engrossing
stories about the early history of the field and the personalities
involved. No mention of his remarkable ability as a teacher would be
complete without reference to the seminars and lectures at which he
imparted scientific knowledge and also entertained his audience as an
incomparable raconteur. His research interests included the
intermediary metabolism of amino acids, creatine, uric acid, and
related compounds, and he was renowned for the discovery, isolation,
and identification of a new amino acid as
When I arrived in Urbana to undertake graduate study in 1943, the
identity of the 10 amino acids essential for growth in rats and the 8 essential for the maintenance of nitrogen equilibrium in the human
(that is, male graduate students) was already known (3, 4). It fell my
lot to isolate, purify, and analyze amino acids and then feed them to
fellow students enlisted as human guinea pigs in experiments involving
daily nitrogen balance determinations. The diets consisted of the
mixture of amino acids under study, the known vitamins, cornstarch,
corn oil, sucrose, butter fat, inorganic salts, and Celluflour (a
product providing roughage but no nutritive value, nitrogen, or
flavor). The only taste thrill in this otherwise bland fare was a large
brown "candy" containing a bitter liver extract as a possible
source of unknown vitamins flavored with peppermint oil and sweetened
with sugar. In those days the recruits were grateful for the free
rations, the dollar a day they were paid, and the prospect of seeing
their initials in print in Rose's widely read publications. The
resulting papers established the quantitative requirements for the
essential amino acids, the availability of some of the D-isomers or
N-acetyl derivatives, and the role of cysteine and tyrosine
in sparing methionine and phenylalanine, respectively. The morale of
the subjects was maintained over many weeks by the prospect of
collecting data for doctoral theses, the obvious importance of our
findings for human welfare, and the infectious enthusiasm of Dr. Rose.
An added benefit in my case was that, while consuming these daily
rations, I had ample time to think about experiments on the metabolism
of the essential amino acids I might pursue later in my career, as
described below.
Rose's students were somewhat in awe of the professor, perhaps
wondering whether they could meet his exacting standards or hope to
emulate the seeming ease with which he succeeded in all of his
professional endeavors. They learned in time that behind his somewhat
reserved and formal manner was a genuine warmth and an understanding
that young scientists develop their full potential only by profiting
from their mistakes. His research achievements earned him wide
recognition and many honors. On the occasion of his 90th birthday his
former students, colleagues, and friends assembled in Urbana to join
him in the celebration. He was much surprised when presented with a
handsome bronze plaque announcing the establishment of the William C. Rose Award and Lectureship. As indicated in Fig.
1, the plaque to be given to all awardees shows his likeness and a sketch of the Noyes Laboratory with the structures of the essential amino acids and the stereochemistry and
crystal structure of threonine, with a quotation and chart from his
classical 1935 paper published in the Journal of Biological Chemistry (2). This award, now administered by the American Society for Biochemistry and Molecular Biology, has been given annually, and the lectures are presented at the Society's national meetings. Until his death at age 98, Will Rose took a keen interest in
those selected for the award named for him. He and his wife Zula
exerted a wonderfully positive influence on all who knew them and took
a personal interest in the 90 graduate students who studied under him,
of whom 56 received the Ph.D. degree. In later years he often commented
on his happy family life until his wife's death in 1965, his exciting
professional life, and the thrill of watching his students grow into
professional stature.
In the first paper of this series on Reflections, Arthur
Kornberg (5) has written perceptively of his deep admiration for Severo
Ochoa, whom he knew particularly well from his stay in that laboratory
in 1946 and their close friendship until Severo's death in 1993. Accordingly, I will comment only briefly on my exposure to that
exciting New York University laboratory during the year 1952. After a
few years as a junior faculty member at the University of Pennsylvania
I had not yet earned an official sabbatical but came to realize that a
knowledge of enzymology was crucial to further progress in my studies
on amino acid metabolism. My colleague Jack Buchanan at the University
of Pennsylvania advised me that the Ochoa laboratory was possibly the
world's finest in enzymology at that time, and I acted on that sound
advice and was most fortunate to have an acceptance. My research there
involved studying the details of acetoacetate synthesis and breakdown
and led to the purification and characterization of coenzyme A
transferase, now called acetoacetyl-succinic thiophorase, from heart
muscle (6).
The Ochoa laboratory was crowded and still in the Pharmacology
Department in an old building on First Avenue, with limited equipment,
including the single Beckman DU spectrophotometer mentioned by
Kornberg. Nevertheless it was an exciting place to pursue research, with Severo's ever optimistic support, intense lunchtime and afternoon discussions that included such luminaries as Otto Loewi, Ephraim Racker, and occasionally Sarah Ratner, and a legion of visiting postdoctoral fellows, present and former students, and sabbatical guests from every corner of the world. Combined with an ethic of
unremitting experimental work, the environment was ideal for a visitor
to master enzymology as an essential tool in understanding carbohydrate
and lipid metabolism.
Unlike other scientific departments I had been exposed to at American
universities, Ochoa's department was more in the European or Japanese
tradition of a group revolving around "the professor." Ochoa (shown
in Fig. 2) was ambitious and inspiring,
exceptionally well informed, and completely dedicated to science. In
describing his career in Europe and the United States (8), he stated
that biochemistry had been his "only and real hobby," but he
greatly appreciated art and music and fully enjoyed their availability in New York City. In the laboratory he talked only of science, but
under more relaxed circumstances his very broad cultural interests came
to the fore.
Because of my increasing interest in mechanistic aspects of
enzyme action, I subsequently took advantage of a sabbatical leave to
improve my knowledge of organic chemistry and spent 1961-1962 with
Professor Vladimir Prelog, Director of the Organic Chemistry Laboratory
of the Eidgenössische Technische Hochschule (Swiss Federal
Institute of Technology) in Zürich. Widely known for his studies
on natural products and his outstanding contributions to
stereochemistry (9), Prelog had developed an interest in enzyme
stereoselectivity, and I began working on oxidoreductases in
Curvularia falcata. The goal was to establish the absolute stereochemical course of hydride transfer to carbonyl groups of substrates such as decalin-1-one, decalin-2-one, and
decalin-1,4-diones. Of interest, all the stereogenic carbon atoms
formed by microbial reduction possess the same
S-configuration, independent of the configuration of the
other stereogenic centers in the molecule or whether the hydroxyls are
in the axial or the equatorial positions. Two Curvularia
enzymes, one believed to favor transfer of hydrogen into the axial
position and another to favor the equatorial position, proved to be
very difficult to purify. We found, however, an enzyme with
oxidoreductase activity toward alicyclic ketones in pig liver that
could be purified and was shown to be the 3-oxoacyl-acyl carrier
protein reductase component of a fatty acid synthetase (10).
Vlado Prelog (see Fig. 3) was a
frequent visitor to the United States and was elected as a Foreign
Associate of the National Academy of Sciences. He said that he
preferred the academic system in which scientific departments had a
number of independent full professors. After he succeeded the famous
Leopold Ruzicka in 1957 at the Eidgenössische Technische
Hochschule, he established a "collegiate leadership" in which all
appointed professors participated, surely an unusual arrangement at
that time in Continental Europe. This gave him more time for research,
for which he received innumerable honors, culminating in the 1975 Nobel
Prize in Chemistry, which he shared with John Cornforth. After his
mandatory retirement the following year, Prelog was required to have
the title of postdoctoral student (Fachhörer) to continue his
work, thus eventually leading to his autobiography entitled "My 137 Semesters of Chemistry Studies" (11).
In addition to his legendary pleasure in scientific study, he was
widely known for his charming and witty personality. I can hardly
recall a meeting with him, even a research conference, where he didn't
regale us with his never ending supply of anecdotes about almost every
famous chemist or biochemist (including those from the past), jokes,
and humorous comments about the shortcomings of totalitarian political
regimes, which he deplored.
In the fall of 1947 I had joined the faculty of the Department
of Physiological Chemistry at the University of Pennsylvania, where I
undertook studies on amino acid metabolism. The department was one of
the first in this country to work with radioactive carbon-14 as a
tracer in intermediary metabolism, and several of the senior faculty
were widely known for their studies on this subject: Jack Buchanan on
purine biosynthesis, D. Wright Wilson on pyrimidine biosynthesis, and
Samuel Gurin on fatty acid oxidation. So little was known about amino
acid metabolism in general at that time that it was difficult to make a
specific choice, but I was intrigued by the branched chain compounds
leucine, isoleucine, and valine. The main reason was that their
metabolic fates might throw some light on the origin of the branched
carbon structures of numerous biologically occurring compounds,
including steroids, vitamins A, E, and K, and a variety of products in
plants, thought to be derived from five-carbon units according to the
biogenetic isoprene rule (12). Another reason to study leucine in
particular was its known ketogenic property (acetoacetate production)
in animals, because the intermediate thought to be formed by
deamination and oxidative decarboxylation, isovaleric acid, was
obviously blocked in the I undertook the chemical synthesis of the substrates labeled with
radioactivity in specific positions, no easy task because 14C-labeled barium carbonate was the only commercially
available starting material, incubated the purified compounds with
liver slices, and analyzed the acetoacetate formed. To my surprise, the
isopropyl group of leucine (and isovaleric acid) provided the terminal
three carbons of this product, but the carboxyl carbon was unaccounted
for. Radioactive CO2 was then employed in other experiments
and found to provide the missing carbon atom. These results (13, 14)
and subsequent experiments with heart extracts (15) thus led to the
discovery of a new ATP-dependent carbon dioxide fixation in
mammalian metabolism.
The scheme in Fig. 4 shows our knowledge
of leucine metabolism as we became aware of the role of coenzyme A and
identified the involvement of the After moving to a faculty position at the University of
Michigan and then returning from sabbatical leave in the Prelog
laboratory some years later, I decided to work on the oxidation of
hydrocarbons, which (because of their poor chemical reactivity) might
be an even greater challenge than leucine for enzymatic degradation. James Baptist, a postdoctoral associate from Illinois, agreed to
undertake this problem and set about isolating a suitable bacterium from soil samples by an enrichment culture technique with hexane as the
carbon source (19). The organism eventually obtained, a strain of
Pseudomonas oleovorans that was dubbed the "gasoline bug" by our colleagues, grew well on several straight chain alkanes (or on leucine) but not on cyclohexane or methylbutane. Cell-free extracts were obtained that required the addition of NADH for the
aerobic conversion of radioactive octane to octanol (20). Thus, it was
evident that alkane oxidation at a terminal methyl group involved
oxygenation as the initial step rather than an ATP-dependent carboxylation reaction, as in leucine
metabolism. We subsequently found that, when presented with fatty acids
as substrates, the bacterial system preferred to attack the terminal methyl carbon atom to give the By preferential extraction of the bacterial cells and column
chromatography, three enzyme components were separated and found to be
required for the conversion of octane to octanol or of laurate to
Verkade et al. (27) in the Netherlands discovered
Then, almost 10 years later, Anthony Lu joined our research group as a
postdoctoral associate after completion of his graduate studies at the
University of North Carolina. He impressed me as a highly talented and
enthusiastic young scientist who would welcome a challenging problem,
and I suggested that we again attempt to characterize the fatty acid
REFLECTIONS
Enzyme Ingenuity in Biological Oxidations: a Trail Leading to
Cytochrome P450
INTRODUCTION
William C. Rose
-amino-
-hydroxy-n-butyric acid, which he named
threonine (2). This was the culmination of experiments in which rats failed to grow on diets containing the 19 previously known amino acids.
Thus, painstaking efforts over many years led to the missing growth
factor found in proteins and in hydrolysate fractions therefrom.
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Fig. 1.
Inscription on bronze plaque given
annually to recipients of the William C. Rose Award in
Biochemistry. The inscription directly below Dr. Rose's portrait
is an excerpt from an article that appeared in 1935 in The
Journal of Biological Chemistry (J. Biol. Chem.
112, 283). It reads as follows: "The data demonstrate
conclusively that the crystalline compound is the new essential we have
been endeavoring to isolate for several years. Furthermore, the
experiments recorded in Chart 1 represent the first successful efforts
to induce growth in animals upon diets carrying synthetic mixtures of
highly purified amino acids in place of proteins." This work marks
the discovery of threonine.
Severo Ochoa
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Fig. 2.
Photograph of Severo Ochoa taken
from the New York Times
(7).
Vladimir Prelog
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Fig. 3.
Vladimir Prelog at
Bürgenstock in 1989 (11).
Branched Chain Amino Acid Oxidation
-position from the entry of a carbonyl
group and, therefore, could not undergo the classical -oxidation
that occurs with straight chain fatty acids.
-hydroxy--methylglutaryl-CoA
cleavage enzyme in generating acetoacetate (16). We had originally
thought from experiments with crude enzyme preparations that the
substrate in the carboxylation reaction was -hydroxyisovaleryl-CoA,
but it was later correctly identified as the unsaturated compound -methylcrotonyl-CoA (17, 18). In summary, we found that nature had
solved the problem of a difficult oxidative reaction by introduction of
an energy-dependent carboxylation as a crucial step. Of
additional interest, our results showed how leucine metabolism is
integrated into the main pathways of lipid metabolism and steroid
biosynthesis. As is well known, the details of cholesterol biosynthesis
were elegantly elucidated by Konrad Bloch and Feodor Lynen.
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Fig. 4.
Metabolic steps in the conversion of
leucine to acetoacetate, with the radioactive labeling pattern obtained
from the methyl carbons ( 
) and 
carbon
(
) of the amino acid and from CO2 (*).
Hydrocarbon Oxidation by Gasoline Bugs
-hydroxy acids (21) rather than to
utilize a more chemically feasible - or -oxidation pathway. More
will be said about -oxidation below in connection with related mammalian enzyme systems.
-hydroxylaurate in the reconstituted enzyme system (22) (Fig.
5). These were purified to homogeneity
and characterized as follows: a red, nonheme iron protein containing
two iron atoms but no labile sulfide and identified spectrally as
rubredoxin (23), previously found only in anaerobes; a flavoprotein
containing one molecule of FAD and found to be the NADH-rubredoxin
reductase (24); and the -hydroxylase, which was relatively insoluble in that it formed aggregates of very high molecular weight, had an
indistinct spectrum, and lost activity upon dialysis that was restored
by the addition of ferrous ions (25, 26). The instability and other
properties of the hydroxylase made it a difficult candidate for
detailed mechanistic studies, but this enzyme system in P. oleovorans has continued to be investigated by others.
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Fig. 5.
Conversion of octane to octanol or of
laurate to -hydroxylaurate in the
reconstituted enzyme system from P. oleovorans under
aerobic conditions in the presence of NADH.
Cytochrome P450: Solubilization, Resolution, and
Reconstitution of the Enzyme System from Liver Microsomal Membranes
Active in Fatty Acid
-Oxidation-oxidation when they fed fatty acids of intermediate chain length
(or their esters or glycerides) to dogs and human subjects and isolated the resulting urinary dicarboxylic acids, and Carter (28) and Bergström et al. (29) later reported that some -
and -substituted fatty acids undergo a similar attack in animals. We
undertook a study to determine the enzymatic mechanism of this
intriguing oxidative process in the late 1950s, but the instability and
insolubility of the liver microsomal enzyme system prevented further
progress, and we turned our attention to the more tractable bacterial
system, as described above.
-hydroxylating enzyme system of liver microsomes, making use of what
we had learned about the bacterial system. The rest of this story is
now well known. The hepatic system was resistant to the isolation and
purification methods employed with the pseudomonad, but fortunately, we
were not discouraged by the lack of knowledge at that time about
membrane-bound enzymes in general and microsomal enzymes in particular.
Thanks to Anthony's painstaking efforts for more than 2 years, the
hydroxylating system eventually yielded to solubilization with various
detergents in the presence of agents to protect against enzyme
denaturation by the detergents. Column chromatography of the resulting
preparations (again with detergents and protective agents) yielded a
red fraction containing cytochrome P450 (identified by the spectral
change upon addition of carbon monoxide to the reduced protein), a
yellow fraction containing the flavoprotein NADPH-cytochrome P450
reductase (assayed by reduction of cytochrome c as an
artificial electron acceptor), and a colorless, heat-stable fraction
(30, 31). The last of these was shown by Henry Strobel, another
postdoctoral associate from North Carolina, to contain phospholipids,
of which phosphatidylcholine was especially active (32). When mixed and incubated together under precise conditions, the three components yielded a reconstituted enzyme system that converted lauric acid to
-hydroxylauric acid in the presence of NADPH and oxygen, as shown in
Fig. 6 (31).
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Fig. 6.
Conversion of any of a variety of
substrates (RH) to products (ROH) in the P450-containing reconstituted
enzyme system from liver microsomes under aerobic conditions in the
presence of NADPH.
TOP
INTRODUCTION
William C. RoseIn the progress of science we all build on previous findings, and we
had the benefit of knowing that microsomes contain a carbon
monoxide-binding pigment of unknown function (33-35), which was
identified as a hemeprotein and designated "P-450" by Omura and
Sato (36). Furthermore, the groundbreaking work of Omura, Sato, Cooper,
Rosenthal, and Estabrook (37) had shown by photochemical action
spectroscopy that this pigment in hepatic microsomes is responsible for
the hydroxylation of several steroids and drugs. Thus, we had in our
hands the solubilized hemeprotein P450 from rabbit liver
microsomes capable of oxidizing not only fatty acids at the terminal
position but a huge variety of other substrates of much greater
biochemical and pharmacological interest. The same methods led to the
successful solubilization and resolution of the P450-containing enzyme
system of human liver microsomes (38). In addition, a visitor from
France, Jean-Michel Lebeault, brought a strain of Candida
tropicalis to my laboratory, and we found that, when grown on the
long chain hydrocarbon tetradecane, it produced cytochrome P450 as the
lauric acid -oxygenating catalyst that could be solubilized and
reconstituted into a functional enzyme system (39). His company was
investigating the use of petroleum as a source of yeast protein for
human consumption, and on a visit to Marseilles I was offered a
"yeast protein burger" that was devoid of the color and odor of the
crude petroleum on which the organism had been grown. Although
undoubtedly nutritious, the product was relatively bland in flavor as
compared with the taste thrill of peppermint-flavored liver I recalled
from my previous experience with nutrition experiments in graduate
school. In the discussion that follows, subsequent work in our
laboratory on catalytic and mechanistic studies with the microsomal
system is briefly summarized. This has become an enormous field of
endeavor, and no attempt will be made in these reflections to provide a comprehensive review. Mention should be made, however, of the seminal
contributions of Gunsalus and colleagues with P450cam, a soluble
(non-membrane-bound) cytochrome from a bacterial source, which is
highly specific for camphor hydroxylation (40).
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Cytochrome P450: Purification and Characterization of Multiple Isoforms |
|---|
Studies with the purified components of the liver microsomal enzyme system have provided insights into structure, function, regulation, and mechanism that would not have been possible with microsomal membranes (41). Predictions from studies with intact microsomes varied from a single P450 enzyme with very broad specificity to an almost unlimited number, each specific for a different low molecular weight foreign compound, just as an antibody is specific for an individual foreign macromolecule. The induced synthesis of drug-metabolizing enzymes supported the existence of several discrete P450s (42), and purification and characterization clearly established the occurrence of a large family of distinct enzymes, including many that individually have numerous substrates (41). These are called isozymes even though that term was coined to describe multiple forms of an enzyme differing in properties such as substrate affinity, maximum activity, or regulation but identical in function. What is now called the P450 superfamily includes members with many different functions.
It took over 5 years for methods to be developed, including column
chromatography in the presence of detergents, for the first mammalian
P450, the phenobarbital-inducible form, to be purified and thoroughly
characterized (43). It was called P450LM2 (liver microsomal
form 2), and shortly thereafter several other distinct forms, including
-naphthoflavone-inducible P450LM4, were purified and
shown to differ in physical and catalytic properties (44), leaving no
doubt that multiple P450 isoforms occur in microsomes. Further
convincing evidence for multiplicity was provided by the differences in
amino acid composition and COOH-terminal amino acid residues (45).
Subsequently still more P450s were identified, including the ethanol-inducible form from liver (46) and unique forms from nasal microsomes (47), as well as numerous other forms from a variety of species, tissues, and organelles by many investigators. The nomenclature became increasingly difficult to follow, sometimes including names of inducers or of any of a variety of substrates, and under the leadership of Daniel Nebert (48) a system was devised based on divergent evolution as judged by sequence similarity. For example, P450LM2 became P450 2B4 and the corresponding gene became CYP2B4 to indicate family 2, subfamily B, and individual enzyme (or gene) number 4. Fortunately, NADPH-cytochrome P450 reductase, the enzyme that transfers electrons to the heme iron atom of P450 and was first purified by Janice Vermilion and then shown to have separate roles in the FAD and FMN cofactors (49), exists as a single form.
Knowledge of the P450 superfamily is expanding rapidly, and it is
evident that this cytochrome occurs throughout nature, including bacteria, fungi, plants, and animals (50). Of particular interest to
the biomedical field, the human species has about 60 functional P450
genes. Detailed analysis of the human genes will be needed to identify
and characterize the complete set of polymorphisms (51) and
disease-causing mutations.
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Cytochrome P450: Catalysis of Multiple Reactions with Innumerable Substrates |
|---|
Considering the rapid progress made in recent years on the characterization of many isoforms, it may seem surprising that P450, a name first used for a red pigment having a reduced CO-difference spectrum with a major band at an unusually long wavelength (about 450 nm), has not been changed to a terminology based on function. Even the term cytochrome is unsuitable, because P450 usually acts as an oxygenase rather than simply as an electron carrier. Since many of the individual P450s catalyze multiple reactions, the usual method of naming enzymes is inadequate for these hemeproteins. The name "diversozymes" has been suggested for P450s, because they are unmatched in the broad scope of their functions (52).
The diverse reactions catalyzed include aliphatic and aromatic
hydroxylation, N-oxidation, sulfoxidation, epoxidation,
oxidative ester and amide cleavage, N-, S-, and
O-dealkylation, peroxidation, ipso-substitution,
deamination, desulfuration, and dehalogenation, as well as reactions
such as reduction of azo groups, nitro groups, N-oxides, and
epoxides that involve only electron transfer and partially justify the
term cytochrome for this enzyme. Additional reactions attributable to
P450 continue to be discovered (53). Two of these, one reductive and
one oxidative, and both believed to involve radical chemistry will be
mentioned. Cumene hydroperoxide was found to undergo reductive cleavage
in the reconstituted enzyme system containing P450 and the reductase in
the presence of NADPH; acetophenone was formed, and the missing
one-carbon product was identified as methane by gas
chromatographic/mass spectrometric analysis. As shown in Fig.
7A, the study was extended to
the 13-hydroperoxide of linoleic acid, which yields pentane and an
aldehyde acid (54). Lipid peroxidation is generally looked on as a
destructive process in membranes of living cells, but molecular oxygen
plays no role in the reductive reaction shown. It may be noted that the
exhalation of pentane and other hydrocarbons by various species,
including the human, is believed to be a measure of this
pathophysiological process, now known to involve P450. Another
P450-catalyzed reaction of much interest is the oxidative demethylation
that accompanies steroid aromatization, for which a role for an
oxygen-derived peroxide has been suggested (55). We have examined the
deformylation of a variety of aldehydes and have proposed a
peroxyhemiacetal-like adduct as a transient enzyme-bound intermediate
(56) as shown in Fig. 7B. Presumably the intermediate
rearranges by a concerted or sequential -scission to yield the
products, formic acid from the aldehyde group and an olefin, an
alcohol, or an alkane from the remaining structure. This reaction is
particularly relevant to the mechanism of oxygen activation by this
versatile catalyst, as described below.
|
The number of organic compounds that serve as P450 substrates was
cautiously estimated in the 1980s to be in the hundreds or even the
thousands, but currently no one familiar with the field is surprised at
the prediction of a million or more. These include physiologically
occurring compounds such as fatty acids, steroids, eicosanoids, lipid
hydroperoxides, retinoids, and amino acids. Equally unexpected is the
very large list of xenobiotic substrates, including almost all drugs
(with many more being produced each year by the pharmaceutical
industry), procarcinogens (57), antioxidants, solvents, anesthetics,
dyes, pesticides, petroleum products, alcohols, and products derived
from plants such as flavorants and odorants. With respect to the
metabolism of drugs, most are inactivated by P450, but some are
activated and others yield products that inactivate the cytochrome
itself (58). Such information is therefore useful for the design and
development of potential new drugs. The ability of this catalyst to
metabolize a multitude of organic compounds that can now be produced
readily by combinatorial techniques but do not occur naturally on this
planet indicates that the number and variety of P450 substrates are
almost unlimited. However, the oxygenation of substrates by the
mammalian cytochromes is not necessarily indiscriminate. Particularly
with compounds of physiological importance, the attack on the substrate
can be both positionally and stereochemically specific.
| |
Cytochrome P450: Mechanism of Action and Evidence for Multiple Forms of Activated Oxygen |
|---|
The mechanistic details of enzyme-catalyzed hydroxylation
reactions have long been of interest, and biochemists and chemists have
been particularly intrigued by the possibility that P450 generates an
unusually powerful species capable of oxidizing relatively inert
substrates. The availability of the purified microsomal cytochromes and
versatility of these catalysts in the metabolism of almost any organic
compound of mechanistic interest helped facilitate rapid progress. For
example, John Groves, a former member of our chemistry faculty,
suggested a collaboration with Ronald White and me on norbornane
oxidation. Sure enough, the exo-tetradeuterated compound was
hydroxylated by P450 2B4; the results suggested an initial hydrogen
abstraction to give a carbon radical intermediate (59). Furthermore,
P450 has also been found to be versatile with respect to the oxidant,
for NADPH, the reductase, and molecular oxygen could be replaced by
hydrogen peroxide (60) and, surprisingly, also by almost any
substituted cumene hydroperoxide, benzyl hydroperoxide, or perbenzoic
acid (61). The accompanying scheme (Fig.
8) indicates the main contributions from
this and other laboratories (62). The identification of oxene,
(Fe-O)3+, as the ultimate oxidant remains elusive, but
mounting evidence is now available that multiple oxidants are involved
in P450 function.
|
In the last few years we have carried out site-directed mutagenesis of
mammalian P450s 2B4 and 2E1 in which the active site threonine was
substituted by alanine to learn more about the details of oxygen
activation. In so doing we have taken advantage of evidence from other
investigators that the corresponding mutation in bacterial P450cam
interferes with the conversion of dioxygen to the oxenoid species by
disrupting proton delivery to the active site (63, 64). Our results
with the truncated, heterologously expressed enzymes (65) support the
involvement of three functional species produced during the reduction
of oxygen (66-68) as shown in Fig. 9.
The occurrence of multiple oxidizing species may contribute to the
remarkable versatility of the P450 family of isozymes in the
modification of drugs and other substrates (69). Furthermore, highly
reactive "radical clocks" employed as mechanistic probes have
confirmed that two distinct electrophilic oxidants effect hydroxylation in cytochrome P450-catalyzed reactions (70, 71). A
related long standing question is whether the thiolate supplied by a
cysteine residue as the proximal heme ligand contributes to the
chemical reactivity of these catalysts. Replacement of the active site
cysteine-436 by serine has recently been shown to convert P450 2B4 into
an NADPH oxidase with negligible monooxygenase activity (72).
|
Many colleagues, including students, postdoctoral associates, and other
collaborators have contributed to the progress of our research.
Regretfully, not all could be adequately recognized in this brief
presentation. In addition, I have benefited from friendships and
interactions with many others in what has become the vast P450
field
so vast, in fact, that the present number of investigators, like
that of the isoforms and their substrates, may be unmatched.
| |
FOOTNOTES |
|---|
Published, JBC Papers in Press, June 5, 2002, DOI 10.1074/jbc.R200015200
Address correspondence to: mjcoon{at}umich.edu.
| |
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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