THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 287, No. 19, Issue of May 4, pp. 15798–15800, 2012
`Published in U.S.A.
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`Classics
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`Author’s Choice
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`Human Cytochrome P450s: The Work of Frederick Peter
`Guengerich
`Purification and Characterization of the Human Liver Cytochromes P-450 In-
`volved in Debrisoquine 4-Hydroxylation and Phenacetin O-Deethylation, Two Pro-
`totypes for Genetic Polymorphism in Oxidative Drug Metabolism
`
`(Distlerath, L. M., Reilly, P. E. B., Martin, M. V., Davis, G. G., Wilkinson, G. R., and
`Guengerich, F. P. (1985) J. Biol. Chem. 260, 9057–9067)
`
`Human Liver Microsomal Cytochrome P-450 Mephenytoin 4-Hydroxylase, a Proto-
`type of Genetic Polymorphism in Oxidative Drug Metabolism. Purification and
`Characterization of Two Similar Forms Involved in the Reaction
`
`(Shimada, T., Misono, K. S., and Guengerich, F. P. (1986) J. Biol. Chem. 261,
`909–921)
`
`Characterization of Rat and Human Liver Microsomal Cytochrome P-450 Forms
`Involved in Nifedipine Oxidation, a Prototype for Genetic Polymorphism in Oxi-
`dative Drug Metabolism
`
`(Guengerich, F. P., Martin, M. V., Beaune,
`P. H., Kremers, P., Wolff, T., and Waxman,
`D. J. (1986) J. Biol. Chem. 261, 5051–5060)
`In the 1980s, the group of Frederick Peter
`Guengerich at Vanderbilt University published
`three papers in the Journal of Biological Chem-
`istry that had a major impact on the pharmaceu-
`tical industry and the field of biochemical re-
`search on cytochrome P450s. The three papers
`described the purification and characterization of
`four cytochrome P450s that metabolized specific
`drugs in the human liver. “Fred Guengerich was
`really the pioneer in understanding human
`P450s,” states Allan Conney at Rutgers Univer-
`sity. The isolation of human cytochrome P450s by
`the Guengerich group introduced a way for the
`pharmaceutical industry to test drugs for human
`toxicity before they are developed and released
`into the market.
`Cytochrome P450s are heme-containing en-
`zymes that function mainly in the liver but are
`also present in other organs. Their job is to oxi-
`dize drugs, toxic chemicals, and endogenous mol-
`ecules such as steroids. Seventy-five percent of
`the enzymes that break down drugs in the human
`body are cytochrome P450s. Five cytochrome
`P450s carry out 90% of the drug breakdown. Four
`of these five cytochrome P450s were described in this trio of papers.
`Guengerich entered the world of cytochrome P450s as a postdoctoral fellow in Minor J.
`Coon’s laboratory at University of Michigan in 1973. “I’ve never gotten out of the business
`since,” remarks Guengerich. “When I got into the game, there were some people who thought
`
`Fred Guengerich of Vanderbilt University.
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`Author’s Choice—Final version full access.
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`there was only one form of cytochrome P450 in an animal. Nobody really knew anything about
`P450s in humans.”
`Researchers in the 1970s, like those in Conney’s group, started to separate multiple forms
`of cytochrome P450 from animals, such as rats and rabbits, demonstrating that, on the
`contrary, an animal could have different types of cytochrome P450. When he established his
`own laboratory at Vanderbilt University in 1975, Guengerich decided to work on rat cyto-
`chrome P450s. By 1982, Guengerich’s laboratory had purified nine different rat cytochrome
`P450s.
`In the early 1980s, approximately 40% of drug candidates failed on the market because their
`pharmacokinetic properties were poorly appreciated in humans. “The whole area of drug
`metabolism in people was really pretty mysterious at that time,” says Guengerich. The
`pharmaceutical industry was testing drugs in animals prior to market release, but when
`people took the drugs, there were often some nasty surprises.
`It was becoming increasingly clear to Guengerich that the human versions had to be
`different from animal cytochrome P450s and warranted their own study. He also knew about
`a clinical pharmacologist named Robert Smith at St. Mary’s Hospital Medical School in
`London who, along with other volunteers, had swallowed 40 mg of a hypertension drug called
`debrisoquine in 1975. Although the other volunteers experienced no side effects, Smith’s blood
`pressure dropped precipitously and remained that way for two days. Later, Smith repeated the
`experiment in families and identified ones whose members were unable to properly process the
`drug. “This may not sound like a big deal, but it really struck me. Basically, [Smith] found that
`some people were missing the gene related to the metabolism of a particular drug,” says
`Guengerich. “This meant there was a single P450 that was dominant in the metabolism of a
`single drug.”
`So in the early 1980s, Guengerich’s group began to focus on purifying the human cytochrome
`P450 that was responsible for metabolizing debrisoquine. However, obtaining good quality
`human livers was a major stumbling block, and the Guengerich group struggled with livers
`obtained from autopsies. Then came a stroke of luck.
`The wife of one of Guengerich’s postdoctoral fellows, Phil Wang, was a nurse at a local
`Nashville hospital. One day, Luke Skelley of the Nashville Regional Organ Procurement
`Agency was visiting the hospital administrators, and Wang’s wife mentioned to him that her
`husband needed good quality human livers for his research. Skelley contacted Guengerich for
`a collaboration. Anytime the agency found itself stranded with a donor liver for which they
`could not locate a recipient in time, the agency would offer the organ to the laboratory for
`research purposes.
`With the collaboration in place, the research took off. Invariably, the call to collect the
`waiting liver “came in the middle of the night,” says Guengerich. “I took my turn with the rest
`of the people in the laboratory with being on call, cutting the liver up when it came in, and
`putting it away in the freezer.”
`Linda Distlerath, a postdoctoral fellow, took the lead on the first JBC paper that described
`the cytochrome P450 that metabolized debrisoquine. The work was “very laborious for a
`number of reasons,” recounts Guengerich. “All the chromatography had to be done in the
`presence of detergents. The detergents had to be removed from each fraction before we could
`assay for the catalytic activity. We had to use a gas chromatography-mass spectrometry assay
`for testing catalytic activity. But somehow, Linda did it, and she found this protein, which we
`called P450DB because it metabolizes debrisoquine.”
`Subsequently, P450DB became known as P4502D6. The same paper described another P450
`that metabolized phenacetin, an analgesic that is not used these days since the discovery that
`it is carcinogenic in rodents.
`Using the same experimental procedures of chromatographic separation, gel electrophore-
`sis, amino acid composition analysis, immuno-inhibition studies, and steady-state kinetic
`assays, Tsutomu Shimada and others from the Guengerich laboratory went on to purify two
`cytochrome P450s that metabolize the anticonvulsant mephenytoin. The group also isolated
`the cytochrome P450 for the vasodilator nifedipine. Guengerich did the bulk of the work for
`that project, so he was first author on the paper.
`The hard and tedious isolations carried by the Guengerich laboratory were “to a much
`greater degree of purity than many of the earlier attempts,” explains Conney. “Human liver is
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`much harder to work with than rat liver because there are smaller amounts of P450s, and
`you’re not treating the humans with inducers to increase the levels of specific P450s.”
`The laborious experimental procedures done by the Guengerich laboratory to purify the
`cytochrome P450s are no longer done, note Guengerich and Conney, thanks to recombinant
`DNA technology. Researchers can just express the cytochrome P450 of their choice in bacterial
`or eukaryotic cell culture and use the enzyme in an assay.
`Now, there are 57 known human cytochrome P450s, “and a lot of that work was done by
`Guengerich,” says Conney. With cytochrome P450s isolated in test tubes, “you can much more
`critically understand the importance of these different catalytic activities,” he adds.
`With the mephenytoin work, Guengerich says that later research showed that the cyto-
`chrome P450s his group had isolated were only part of the story. “We thought at that time that
`we had the mephenytoin hydroxylase. Later work by others would show that another enzyme
`did that, and we were only having a little bit of activity in our fractions.” As for P450NF, which
`metabolizes nifedipine, now known as P4503A4, “we had no idea that this one would actually
`wind up being the main player and work on half the drugs on the market,” states Guengerich.
`It also turns out P4503A4 and the cytochrome P450 that metabolizes phenacetin, P4501A2,
`play a major role in the bioactivation of carcinogens. “These are major areas of interest in the
`field of cancer research and molecular epidemiology,” says Guengerich. He explains there is a
`possibility that the amount of some of these enzymes a person has will determine whether or
`not he or she is predisposed to cancer when exposed to an environmental carcinogen.
`Guengerich says his body of work clearly illustrates how fundamental research can influ-
`ence applied research. “I’ve been very fortunate that I’ve been able to find a niche where I can
`do fundamental biochemistry,” he says. “I consult a fair amount in the pharmaceutical
`industry, and it’s neat to see some of the things we did actually have a major impact on how
`people develop drugs.”
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`DOI 10.1074/jbc.O112.000003
`
`Rajendrani Mukhopadhyay (ASBMB’s Senior Science Writer) wrote the introduction.
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`Human Cytochrome P450s: The Work of Frederick Peter Guengerich
`2012, 287:15798-15800.
`doi: 10.1074/jbc.O112.000003
`
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