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SUMMING UP

Konrad Bloch

James Bryant Conant Laboratories, Harvard University, Cambridge, Massachusetts

02138

To look ahead, to think of research problems still to be solved, comes more
naturally to most scientists than to reflect on the past. Yet the opportunity to
write a retrospective account like this has not been the chore I expected it to
be. On reflection one sees the Why and the How of one's upbringing and
career more clearly and from a perspective that is missing or has no place in
the professional publication record. That circumstances and good fortune
rather than deliberate decisions on my part have played a major role in my
career is now much clearer to me. To begin at the beginning:

I was born and grew up in Neisse, Germany, in the province of Silesia, the
second child of a middle class family in comfortable circumstances. My father
had studied law but to his regret never practiced it. Instead, as a dutiful son he
took over the family factory, but he never enjoyed being in business. Neisse
was a midsized country town noted for several Gothic and Baroque churches,
a beautiful Renaissance town hall, and a 300-foot-high watchtower. Hidden in
the hills surrounding Neisse were fortifications dating to the era of the
Prussian king Frederick the Second, who annexed Silesia from the Austrian
Empress Maria Theresia. To her, we were told in school, Silesia was the most
precious jewel in her crown. Perhaps for this reason we took little pride in
being Prussians. My upbringing at home was fair and understanding yet strict,
in line with customs and practices of the time. Only in one instance did I come
near to rebelling against parental authority. On the occasion of my Bar
mitzvah a generous great-uncle asked me to choose a present, the choices
being either a cello or a canoe. For a 13-year-old the decision was obvious.
Canoeing would be pure pleasure while practicing a musical instrument meant
hours of drudgery. Thinking otherwise, my parents insisted that it was the
cello I really wanted-and they won. It took many years before I came to

0066-4154/87/0701-0001$02.00

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terms with the cello, but eventually I enjoyed playing it. That the canoe was
really not for me I learned a few years later. After graduating from high
school, I spent the summer of 1930 in a steel works near Lubeck at the Baltic
Sea to obtain the practical experience required for an engineering degree, my
goal at the time. It was a beautiful day when a friend asked me to join him on

a canoe trip. I accepted eagerly but at the end of the day I was burned to a

crisp and had to be hospitalized. Later in this account I will relate a second
incident that bore out the wisdom of the parental decision.

I have only good memories of the schools I attended, in spite of the fact that

most of the gymnasium teachers were martinets, some to the point of brutal­

ity. Learning by rote was considered an important, even essential educational
principle. In retrospect one wonders whether there is a superior one, at least
for adolescents. No doubt the system laid a sound basis for higher education
even if in my case it failed to stimulate any special interests. Surely the

chemistry teacher could not claim to have influenced my career. He suc­

ceeded in making the subject matter totally unattractive. As for my future,
there was no family tradition to follow and therefore no parental pressure to

enter one career or another. There were physicians among my forebears, but

no scientists. I knew next to nothing about academic careers, and Einstein was
probably the only living scientist whose name I knew. As for my interests as
an adolescent, they tended towards the natural sciences or engineering. The

few books I brought along from Europe as sentimental baggage included a set

of slim volumes, published by Sammlung Goschen, on subjects such as

mineralogy, crystallography, stereochemistry, and metallurgy. The last sub­

ject intrigued me most, and led me to read the authoritative textbook of the
time, Chemistry and Physics ofMetals and Their Alloys by Gustav Tamann.
Stainless steel had just been invented, and there was much optimism that the
time had come for a rational metallurgy, the design of alloys with a wide
range of desired properties. I enrolled at the Technische Hochschule in
Munich, but the uninspired course in metallurgy soon dampened my enthu­
siasm for the subject. The chemistry courses were a different matter. I no
longer quite understand why I enjoyed the introductory courses, which

emphasized quantitative and qualitative analysis. Obviously I must have

derived much satisfaction from chemical manipulations and their visual

aspects. Of lasting impact, however, was Hans Fischer's organic chemistry
course. As he presented it, the subject matter was fascinating, the organiza­
tion superb, and the delivery monotonous. To play to the galleries or to raise
his voice for emphasis was not Fischer's style. At the end of my second year I
knew I had found my field. Natural products chemistry then flourished in
Germany, and Hans Fischer was one of the prominent figures in a stellar
group that included Richard Willstaetter, Heinrich Wieland, and Alfred Win­
daus. Their lectures to the "Munchener Chemische Gesellschaft" have re-

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mained vivid memories. I remember especially Wieland's seminar in 1934 on
butterfly pigments. He had discovered these novel structures and named them
pteridines. His lecture ended on an emotional note. Wieland had isolated the
pigments from some 200,000 butterflies schoolchildren all over Germany had
collected for a penny a piece. In concluding, Wieland remarked, "I will be
unable to continue this research since the Government regards the collecting
of butterflies as cruelty to animals, incompatible with the ethics of the
National Socialist party. I can only hope"-he added-"that this research will
be continued by my colleagues abroad." To make this rather mildly critical
statement in front of numerous brown-shirted students took considerable
courage. To me Heinrich Wieland has remained a model, a great human being
as well as a great scientist. The next two years I had to spend essentially full
time in the organic lab. Some 100 "Gatterman" compounds had to be deliv­
ered to the Assistent, and after they passed the test, one advanced to so-called
literature preps that required more skill and stamina. The latter were either
pyrroles (in kg quantities) or porphyrins (in gram quantities) to be passed on
as starting materials for the synthetic work of PhD students. H. Fischer had
just completed the total synthesis of hemin, and chlorophyll was the next
goal. This was perhaps the most physically demanding period in my life. To
give an example, porphyrins were separated by distribution between Hel and
ether in thick-walled and t�erefore heavy lO-liter separatory funnels.

In early 1934 the dean of the Technische Hochschule notified me that I was
ineligible to continue in chemistry since Professor Fischer "had declined to
accept me as a graduate student. " This was a lie but in line with the impending
racial laws. With a naivete that seems incomprehensible in retrospect, I
explored opportunities for doing graduate work elsewhere in Europe. Fortu­
nately I received negative replies to inquiries I had sent to A. Butenandt, then
at the Technische Hochschule of the "Free City of Danzig," and to F. Kogl at
Utrecht. Events moved fast; Danzig was incorporated into the German Reich
a few months later and Holland overrun by the Nazis early in World War II.
To have been denied admission to the Utrecht department proved a lucky
escape for yet another reason. In the mid-1930s the Utrecht laboratory
claimed two spectacular discoveries: 1. "Heteroauxin," supposedly a new
plant hormone, isolated from thousands of liters of horse urine and 2. the
occurrence: of 0 amino acids in tumor proteins. Both "discoveries" proved to
be fabrications by one of K6gl's coworkers. Only after the war had ended did
the truth come out.

Anxious to help me, H. Fischer, an ardent patriot but not a Nazi and
impeccably fair to all of his students, arranged my appointment as research
assistant at the Schweizerisches H6henforschung's Institut in Davos. This
delightful mountain resort had two claims to fame. Its ski runs were the
longest in the Alps, and it had the largest number of tuberculosis sanatoria in

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the world. Thomas Mann had chosen it as the setting for The Magic
Mountain!, his most famous novel.

Frederic Roulet, a pathologist and head of the Institute, directed research
appropriate to the locale. In his enlightened view, the cure for tuberculosis
would ultimately come from biochemical studies, specifically the analysis of
mycobacterial lipids. My predecessor at the Institute claimed to have found
cholesterol among the lipids of human tubercle bacilli, contradicting an earlier
report by Erwin Chargaff (1935). Worried about the reputation of the In­
stitute, Roulet asked me to sort things out. This was my first research
experience, a trying one for a chemistry undergraduate who had no one to tum
to for advice. My inexperience showed during my first day in the laboratory.
Whatever chemical operations one undertook 50 years ago, the first essential
task was to purify all organic solvents. No matter how often I distilled
acetone, I was unable to raise its boiling point above 52°C. Eventually it
dawned on me that at an altitude of 5000 feet this is what it should be and not
56°C. I was now ready to repeat my predecessor's experiments. From lipid
extracts of tubercle bacteria he had been able to obtain an insoluble di­
gitonide, the classical test for cholesterol, and I confirmed him up to this
point. However, pyridine cleavage of the precipitate failed to yield the
expected, easily crystallizable cholesterol. It was apparent that a branched
chain aliphatic hydrocarbon and not cholesterol was responsible for the

'While writing this chapter I happened to take another look at the novel in order to refresh the

memory of my Davos years. This effort was rewarded when I came upon the following prophetic
passage:

"Nothing different.-Oh, well, the stuff to-day was pure chemistry," Joachim un­
willingly condescended to enlighten his cousin. It seemed there was a sort of poisoning, an
auto-infection of the organisms, so Dr. Krokowski said; it was caused by the disintegration
of a substance, of the nature of which we were still ignorant, but which was present

everywhere in the body; and the products of this disintegration operated like an intoxicant

upon the nerve-centres of the spinal cord, with an effect similar to that of certain poisons,
such as morphia, or cocaine, when introduced in the usual way from outside.

"And so you get the hectic flush," said Hans Castorp. "But that's all worth hearing.
What doesn't the man know! He must have simply lapped it up. You just wait, one of these
days he will discover what that substance is that exists everywhere in the body and sets free
the soluble toxins that act like a narcotic on the nervous system; then he will be able to
fuddle us all more than ever. Perhaps in the past they were able to do that very thing. When

I listen to him, I could almost think there is some truth in the old legends about love potions

and the like."

To my surprise none of the Thomas Mann experts nor any of the neurobiologists I consulted

were aware of this remarkable prediction of endorphins and enkephalins-made more than 70
years ago! It was of course pure accident that I reread The Magic Mountain now and not 10 years

earlier, before the discovery of "the body's own opiates." My attempts to find the sources for
Mann's inspiration have not yet yielded any clues.

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insoluble digitonide. Roulet was not happy with the result but was persuaded
to accept my negative findings when an American paper by R. J. Anderson
and R. Schoenheimer confirmed the absence of cholesterol in mycobacteria as
Chargaff had reported earlier. This was my earliest encounter with cholester­
ol. That I would find myself in the same laboratory with Erwin Chargaff at
Columbia a few years later and that R. J. Anderson and Rudolf Schoenheimer
would play crucial roles in my career I had no reason to suspect.

With the cholesterol problem out of the way I was next asked to investigate
the phospholipid chemistry of the same microbes. It was known that the
phospholipid fraction of the human tubercle bacillus, when injected sub­
cutaneously, causes tissue changes (tubercular granulomas and giant cells)
indistinguishable from those elicited by live bacteria. Roulet felt that the
species specifity (human versus bovine) of the purified "phosphatide" frac­
tions was worth investigating. I had not heard of phosphatides before, and
learned everything then known about these substances from a monograph by
Thierfelde:r and Klenk. I soon discovered that R. J. Anderson, professor and
chairman of biochemistry at Yale University, was the foremost authority on
the phospholipids of acid-fast bacteria. To my request for reprints he re­
sponded promptly. I followed his directions for preparing the so-called phos­
phatide A-3 fraction, but distressingly my elementary analyses of this sub­
stance showed a higher phosphorus content and the complete absence of
nitrogen, at variance with Anderson's analytical data ( l , 2).

With aU the deference that seemed proper for a German undergraduate
when writing to a senior professor, I informed Anderson of my results: "Can
you advise me what I might have done wrong?" The reply from Yale was
prompt and what it said astonishing: "Looking over our data, it seems to me
quite possible that your preparation is purer than ours. " Phosphatide A-3
actually proved to be phosphatidic acid2. Apart from boosting my morale,
Anderson's letter gave me the courage to tum to him for help. My residential
permit in Switzerland was about to expire without any chance of extension.
To emigrate to the United States had long been my hope, but had remained a
dream for want of American relatives who might provide the required affida­
vit of support. Again the response from overseas was prompt. Two letters
arrived and the news was rarely, if ever, more welcome. "I have the pleasure
to inform you that you have been appointed Assistant in Biological Chemis­
try, School of Medicine, Yale University." So wrote Michael Winternitz, the

20nly youthful enthusiasm explains that I volunteered to serve as the experimental animal for

testing the biological activity of the two phosphatide fractions. The material from the human
strain was injected into my left and the bovine phosphatide into my right forearm. Only the
human fract!ion gave a positive response. Roulet was greatly pleased and I was left with two-inch

scars still highly visible today. I should add that in my innocence I had paid little attention to the

observation that tubercle bacilli remained viable after repeated washings with acetone.

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dean. Anderson's separate letter was sober: "I hope the Dean's appointment
letter will be of help to you. Unfortunately no funds for a stipend are available
to go with the appointment. " The American consul in Frankfurt-perhaps a
Yale man-issued the immigration visa forthwith. I had not shown him
Anderson's letter.

My morale needed a boost for another reason. In Roulet's opinion the
publications resulting from my work in Davos (1,2) might be sufficient for a
PhD thesis. I enrolled at the University of Basel, and in due time submitted
my thesis. Dekan Professor Bernoulli, a descendant of the famous physicist,
was most encouraging. Yet a month later a curt letter arrived stating that the
Naturwissenschaftliche Fakultaet had unanimously rejected my thesis as "un­
geniigend." While visiting the Basel Biozentrum a few years ago, I could not
refrain from beginning my seminar by commenting on my dismal experience
in 1936. My host's curiosity was aroused. From the university archives he
extracted the information that one member of the faculty committee had
rejected my thesis because I had failed to cite some important references (to
his publications). According to the rules at that time one dissenting vote made
the rejection unanimous. Very much later I wondered whether or not this was
another occasion when fate was kind to me.

I arrived in the United States in December 1936 with great hopes but barely
enough funds to support me for a month. While still in Munich I had not dared
to aspire to an academic career. I expected to end up in the chemical industry
like most of Fischer's students. At the time financial independence was a
prerequisite for a "Habilitation," at least in practice. The Privatdozent, holder
of the lowest academic rank, received no fixed salary and was expected to
subsist on the meager tuition fees paid by the few students who enrolled in the
special topics course a Privatdozent was allowed to teach. The large, remu­
nerative courses were reserved for the professor. The larger the number of
students, the greater the professorial income. Understandably the larger uni­
versities were also the best.

My initial euphoria of having made it to the United States began to subside,
and reality demanded that I support myself. After scanning the ad section of
the New York Times I applied without much enthusiasm for an opening in a
small dye factory in New Jersey. Yet I felt that in spite of my distressing
experience in Basel, I must at the very least explore the possibility of going on
to a PhD degree. In the interim, however, I had not failed to pay my respects
to Rudolph Anderson. Without his help I might not have made it to these
shores. Besides, while I was still in Switzerland, he had offered to take me on
for graduate work. Yet he now discouraged me from coming to Yale: "You
won't learn very much here; why don't you work with Hans Clarke at P & S?"
(College of Physicians and Surgeons, Columbia University). This modesty
hardly conformed with my image of a Herr Professor but it was in keeping
with Anderson's readiness to help some unknown youngster who had earlier

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dared to criticize his experiments, if ever so politely. This and other early
contacts with senior scientists in the United States impressed me deeply and
laid the basis for my undiminished regard and continuing admiration for the
open and democratic spirit that prevails in American academia.

From abroad I had brought with me two letters of recommendation, one
from Richard Willstaetter, whose counsel I had asked before leaving Munich,
and a much shorter but very effective one from Hans Fischer. In essence his
letter said no more than "Herr Bloch ist gut. " Fischer had suggested that I seek
advice from Max Bergmann at what was then the Rockefeller Institute. My
visit to the institute proved to be a crucial tum of events. Bergmann agreed
with Anderson that to work in Hans Clarke's department was my best bet. An
interview was granted forthwith, and when the departmental secretary took
me to his lab, Clarke asked me to wait a minute until he had finished taking a
melting point. Since the compound melted sharply, Clarke was in good
humor. He obviously had a high regard for the training students received in
Hans Fischer's lab, and moreover his sympathies for refugees and his efforts
to help them were widely known. The friendly interview and the probing of
my background ended with the question, "By the way, do you play a musical
instrument?" In good conscience I could answer "yes." I was not asked how
well I played the cello. End of interview and admission as graduate student.
Once again I had reason to be grateful for my parents' foresight. Elated by the
outcome I reported the acceptance to Bergmann, who immediately offered to
find financial support: "I will introduce you to a friend who might help. " The
friend was Leo Wallerstein, head of the Wallerstein Laboratories on Staten
Island, New York, consultants to the brewing industry. An early immigrant
from Gennany, Wallerstein held a lucrative patent for controlling the turbidity
of Lager beer by addition of proteolytic enzymes. His foundation assisted
refugee scholars, beginning as well as established. With financial support
assured for a year I could go to work.

Clarke, unlike the Basel faculty, did not find my published Davos papers
wanting and generously accepted them as partial fulfillment for the PhD
requirements. Still, to satisfy formalities I had to carry out some research at
Columbia, but the problem Clarke assigned to me was straightforward and not
demanding. For my thesis problem I was to synthesize a number of N­
alkylcyste:ine derivatives and to examine their sulfur lability. The work went
smoothly and according to plan except for the failure of N-methylcysteine
hydrochloride to crystallize. Eventually, however, some crystals appeared
and spread steadily. I took the round-bottom flask to Clarke, who smilingly
told me about Adolph von Bayer's habit of expressing his appreciation for a
student's success. On such occasions, when making the rounds, von Bayer
would lift the Calabrese hat he wore indoors and outdoors. Clarke's symbolic
gesture made this a sweet moment. It became even sweeter for the graduate
student when Clarke suggested I call up "Dee" (Vincent du Vigneaud,

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Professor and Chairman of Biochemistry at Cornell) to offer him a seed
crystal-du Vigneaud's preparation of N-methy1cysteine had not yet crystal­
ized. du Vigneaud himself came to l68th Street and was most gracious.
Nowadays a sharp chromatographic peak may be a superior criterion for
purity, but as an aesthetic experience, it does not compare with the sight of
crystals one is the first to see. Only X-ray crystallographers still experience
this visual pleasure. After a year and a half, Clarke decided that along with
my earlier papers, I had sufficient material for a thesis and for publication.
The resulting J. Bioi. Chern. paper (3) was a mere 12 pages long at a time
when the journal format was one half of what it is today, the margins were
wide, and the print at least twice as large.

In 1938 the Depression was still severe, and I found myself with a degree
but without a job in sight. Max Bovamick, a physician working for his Ph.D.
in the same laboratory, needed an organic chemist in order to test his idea that
a diodophenyl derivative of thyroxin should be of interest. A molecule
containing six iodines instead of four, and three phenyl rings instead of two,
was bound to be a superhormone. I managed to synthesize the monster, which
proved to be totally inactive biologically. Its only distinction was complete
insolubility in water. The outcome taught me an early lesson: Avoid chemical
reasoning for predicting biological activity! This interlude came to an end one
day when Rudolf Schoenheimer asked me to join his group. Later he con­
fessed that he had hesitated hiring me because my thesis was so "thin," as
indeed it was. Up to this point I had a modicum of experience in preparative
organic chemistry but my knowledge of biology or biochemistry was nil. I
accepted Schoenheimer's offer at once and soon realized that biochemistry
was going to be my field from now on. How brilliantly Schoenheimer seized
and exploited the opportunity of using stable isotopes for tracing metabolic
pathways is a matter of record. It is tragic that Schoenheimer died too young
to reap the rewards due to him.

Harold Urey's generous donations of stable isotopes and David Ritten­
berg's collaboration played essential roles in this pioneering research. To
develop the tracer methodology required novel or specially adapted synthetic
and analytical procedures and above all the insight to identify problems that
could be solved uniquely with the aid of isotopes. A seminar by Harold Urey
that Schoenheimer attended had convinced him that isotopes were the answer
to a biochemist's prayer, especially for elucidating biosynthetic transforma­
tions. Reactions that are uphill energetically were essentially unknown in the
1930s. Besides, the tracer method was noninvasive and therefore gave in­
formation on metabolic events under normal physiological conditions. Almost
overnight it superseded the classical balance method, which had inherent
limitations even though it had furnished remarkably accurate information in
some instances. Schoenheimer was fond of dramatizing the advance by
comparing the balance method to a Coca-Cola dispenser. Feeding compound

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A to an animal and isolating an increased amount of B from the tissues or
excreta no more proved the conversion of A to B than the observation that a
coin dropped into the vending machine is converted into a bottle of coke. One
balance experiment that should have worked but did not was the conversion of
muscle creatine into urinary creatinine. To settle this issue with the aid of 15N2
was my first assignment. It was straightforward enough except that all earlier
and inconclusive nonisotopic experiments had been done with humans or
dogs. 15N2 was precious and the labeled creatine I synthesized barely suf­
ficient for feeding it to a few rats. But the isolation of creatinine by direct
precipitation with picric acid, which worked so well with human urine, failed
with rat urine. After I had laboriously devised a chromatographic method for
isolating creatinine from this source, the creatine-creatinine conversion was
proven but it was hardly a surprising discovery. More interesting was the next
project, to elucidate the mechanism of creatine synthesis. The question was
whether the methylation step occurred early or late in the pathway. A priori
two mechanisms seemed reasonable, one involving the methylation of glycine
to sarcosine and the other the methylation of guanidoacetic acid. In either
case, the guanido group of creatine might originate from arginine. The
pathway glycine + arginine � guanidoacetic acid � creatine proved to be
the one nature has chosen. H. Borsook and J. W. Dubnoff (4) had already
contributed to this problem by demonstrating the methylation of guanidoace­
tic acid to creatine. In the course of this work we became aware of some
elegant amino acid chemistry M. Bergmann and L. Zervas had described (5).
Twelve years earlier they had observed the transfer of an acetylated amidine
group of arginine to glycine ethyl ester to form guanidoacetic acid ester, a
gratifying model for the enzyme-catalyzed reaction. In this case nature pro­
ceeds "logically," following chemical precedent. Transamination is one ex­
ample of fulfilled expectations and E. Snell's model studies on transamination
another, and perhaps the best known one. Much more often, however, the
choices of nature are not those the chemist would predict. Perhaps for this
reason some chemists of earlier times found the unexpected designs of nature
unsettling and therefore biochemistry without appeal, at least as subject for
their own research. Fortunately this is no longer true, so much so that
chemists have now taken over much of the territory that used to be the
biochemists' preserve.

My first apprenticeship with Rudi and membership in his congenial and
dedicated group were immensely enjoyable and gave me a superb education. I
saw no reason to look for a job elsewhere. Yet when an offer came from Mt.
Sinai Hospital in New York to join a Cancer Research team at twice the
stipend I received at Columbia, I could not resist. I was anxious to marry and
did not want to do so on a minimal budget. The interlude at Mt. Sinai was
brief. After less than a year, Schoenheimer invited me to return to Columbia
and offered to match the Mt. Sinai salary. Fortunately the hospital was

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understanding. On my return to Columbia Schoenheimer suggested that I
investigate the origin of the hydroxyl oxygen in cholesterol. Was water or
molecular oxygen the source? In retrospect, it is truly remarkable that these
two alternatives should have occurred to Schoenheimer. In 1940 oxygen was
widely believed to serve exclusively as a terminal electron acceptor in respira­
tion but not as a source of carbon-bound oxygen in organic compounds. The
much earlier proposal of Bach of peroxidase-catalyzed oxygenations had
fallen into disrepute for lack of experimental evidence. At any rate, I failed to
make progress with this problem because no method existed for the mass
spectrometric analysis of stably bound oxygen in complex organic com­
pounds. I was to develop such a method, but on this as well later occasions I
demonstrated my ineptitude and lack of enthusiasm for designing analytical
procedures. Had I been more skillful, the first oxygenase reaction might have
been encountered some 15 years before Howard Mason and Osamu Hayaishi
independently demonstrated the direct introduction of O2 into aromatic sys­
tems. Their discovery of oxygenases opened one of the most remarkable new
chapters in biochemical research.

Schoenheimer's untimely death in 1941 left his associates without the
leader and the inspired leadership they so admired. We feared that we might
have to look for jobs elsewhere, but Hans Clarke encouraged us to continue as
heirs to the wealth of projects Schoenheimer had begun and developed. From
now on we were to proceed on our own and to choose problems as we saw fit.
This created somewhat of a problem since none of us had proprietary claims
on a given subject. We shared the methodology but had no explicit commit­
ment to any of the major areas then under investigation. How the division
of "spoils" came about I do not recall-it may have been by drawing lots.
At any rate, David Shemin "drew" amino acid metabolism, which led to
his classical work on heme biosynthesis. David Rittenberg was to continue
his interest in protein synthesis and turnover, and lipids were to be my
territory.

At the time I was struggling with the cholesterol oxygen problem but
quickly lost all interest to go on with it. A major turning point came with the
arrival of a paper by Sonderhoff & Thomas (6), which had been delayed by
the war. It reported that "The nonsaponifiable fraction of yeast grown in a
medium supplemented with D-acetate had a Deuterium content so high that a
direct conversion of acetic acid to sterols has to be postulated. " This result
was gratifying to Rittenberg, since his and Schoenheimer's experiments with
020 had indicated that animal cholesterol is synthesized from small mole­
cules, most likely intermediates of fat or carbohydrate metabolism. The
obvious experiment to do, and it was done the next day, was to feed labeled
acetate to rats and mice. Incorporation of deuterium into fatty acids and
cholesterol was substantial, an outcome that set the stage for my long-lasting
interest in the biosynthesis of cholesterol and fatty acids. Gratifying as it was,

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this result did not tell u s how many of the 2 7 sterol carbon atoms were
supplied by acetic acid. Feeding of a labeled metabolite leads necessarily to
isotope dilution of unknown magnitude. Rough estimates of the endogenous
acetate pool suggested, however, that acetate was a major and perhaps
specific carbon and hydrogen source for all moieties of the sterol molecule
although other precursors were not ruled out. The definitive answer came 10
years later with the arrival of microbial mutants. After I had moved to
Chicago, I became aware of an acetateless mutant of Neurospora crassa that
Ed Tatum, then at Yale, has isolated. The mutant seemed ideal for our
purposes. Joining forces with the Yale investigators, I. Zabin could show that
in this mutant, the sterol synthesized derived all its carbon atoms from
exogenous acetate (7). Perhaps this result s hould have stimulated me to tum to
microbes as experimental systems, but for such a drastic change I was not yet
prepared. To establish the origin of individual sterol carbon atoms from either
acetate carboxyl or methyl by traditional chemistry was to take several more
years. This problem and the elucidation of biosynthetic pathways in general
that occupied biochemists in the 1940s and 1950s could have been solved in a
fraction of the time with the technologies available today. Yet the slow pace
of getting there had rewards of its own.

My remaining years at Columbia were spent showing the suspected pre­
cursor role of cholesterol for bile acids and steroid hormones, if proof was
needed. However, structural similarities, no matter how suggestive, do not
prove biochemical relationships. Experience has taught biochemists to
approach all such problems with an open mind. To demonstrate the origin of
progesterone from cholesterol, which I thought needed to be shown, raised
both technical and logistic problems. First of all, to prepare the requisite
labeled cholesterol in sufficient quantity proved to be a major effort. Labeled
compounds of any kind were not available commercially in the early 1940s.
Much time was spent introducing deuterium into cholesterol by platinum­
catalyzed exchange in heavy water-acetic acid mixtures. This method was
destructive, but yielded gram quantities containing 4-5 atom percent D.
Secondly, human pregnancy urine was the only practical source for isola­
ting the progesterone metabolite pregnanediol in sufficient quantity. My re­
quest to the P & S department of obstetrics and gynecology for permission to
administer labeled cholesterol to one of its patients was brusquely denied.
Fortunately, however, I found sympathy and willingness to cooperate at
home. The experiment gave the expected result, but ever since I have felt
somewhat guilty that the essential collaborator was not a coauthor and men­
tioned only anonymously in the experimental section of the 1. Bioi. Chern.
paper (8).

During the war years universities had suspended new appointments and
promotions. Some of my contemporaries and I grew increasingly restive
about the temporary nature of our appointments even though Hans Clarke

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allowed us total freedom in the choice of our research. The first break came
early in 1946 when I was invited to Salt Lake City to be interviewed for an
assistant professorship in the biochemistry department. During the discussion
following my seminar, I rather curtly responded to a comment from the
audience, not realizing that the very youthful questioner was someone very
high up in the administration. I never learned whether my interview went
well, but I suspect it did not. Breaking my return journey in Chicago, I visited
Earl Evans, the recently appointed chairman of the biochemistry department
of the University of Chicago, who was recruiting actively. As graduate
students at Columbia Earl and I had worked at adjacent benches and had
become good friends, sharing tastes in literature and music. Without any
preliminaries Earl asked me whether I was interested in joining his department
as an assistant professor. To accept was an easy decision, even if it meant
sacrificing the prospect of skiing the wide open slopes of the Wasatch
mountains in Utah. To join Evan's department was especially attractive
because an isotope laboratory with a mass spectrometer that functioned more
than half of the time had already been set up by Herbert Anker, my first
graduate student at Columbia.

Once independent, an investigator may choose to play it safe by continuing
earlier, ongoing research or he or she may venture in new directions. The
decision is a matter of temperament and imagination, not necessarily of
intelligence. I decided to temporize and to play it safe, although not entirely.
Sterol biosynthesis, far from being finished, seemed too challenging a prob­
lem to abandon. At the same time, the temptation to branch out and to explore
some other biosynthetic pathways was difficult to resist. In the early 1950s
one of the major unresolved problems that clearly deserved a new look was
protein biosynthesis. Several laboratories approached it, in retrospect naively,
by examining the formation of peptide or amide bonds. Yet the underlying
hypothesis that biosynthetic reactions require energy, specifically ATP,
proved ultimately correct and an important first step. John Speck, my neigh­
bor in Chicago, studied glutamine synthesis from glutamate and ammonia.
Fritz Lipmann, who had earlier proposed that ATP serves as a general energy
carrier for group activation, approached the problem by investigating the
acetylation of sulfanilamide, and thus discovered coenzyme A. I chose glu­
tathione synthesis as a model system and carried out some preliminary
experiments myself. Robert Johnston took on the problem for his graduate
work and could demonstrate that the tripeptide is synthesized in cell-free
extracts and that ATP stimulates the process. Several years of hard and
skillful work by John Snoke then led to the isolation and partial purification of
the two component enzymes for the formation of the tripeptide via '}'­
glutamylcysteine (9). A few years later (1955), when Paul Zamecnik and
Mahlon Hoagland launched the modem era of protein biosynthesis, it became
obvious that apart from the ATP requirement, glutathione synthesis had no

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bearing at all on the mechanism of polypeptide formation. Yet, as my
laboratory's first experience in enzyme purification it was not a wasted effort.
This and other forays were undertaken by a group of unusually talented and
mature graduate students (C. Gilvarg, R. Johnston, I. Zabin, R. Langdon, O.
Reiss) eager to work on problems of their own rather than as members of a
team. In one such project Charles Gilvarg explored the still unknown origin of
the aromatic rings of phenylalanine and tyrosine. Might they originate from
acetic acid like the cyclohexane rings of sterols? This proved not to be the
case. Instead, the isotopic data seemed compatible with a "condensation of a
triose unit with products of glucose metabolism leading possibly to a 7 carbon
sugar" (10). When B. Davis discovered shikimic acid as an intermediate in
aromatic biosynthesis (195 1) with the aid of Escherichia coli mutants our
traditional approach seemed obsolete. Gilvarg saw the light and joined B.
Davis's laboratory contributing to the classical investigations that elucidated
the biosynthesis of aromatic amino acids. In the meanwhile studies on the
mechanism of cholesterol biosynthesis continued but progressed slowly. The
goal was to establish the origin of each of the 27 individual carbon atoms of
cholesterol, from acctatc mcthyl or acetate carboxyl, respectively. This
formidable: task was eventually completed by parallel and complimentary
efforts in the laboratories of Cornforth and Popjak and our own (11, 12).
Happily, it was a friendly, agreed-upon division of labor, not a race. But the
road from acetate to sterol was obviously long, and the chemistry unpredict­
able. Few if any of the hypotheses that steroid chemists had proposed over the
years gave: useful guidance or were experimentally testable. Yet one of the
clues and :;peculative ideas, not widely quoted, seemed worth pursuing. My
next door laboratory neighbor Thomas Gallagher reminded me of a report by
the British nutritionist H. J. Channon showing that feeding of the shark oil
hydrocarbon squalene increases the cholesterol content of animal tissues (13).
To Channon the chemical structures of neither precursor nor presumed prod­
uct were known and his evidence based on balance experiments suggestive at
best. However, his paper had the virtue of stimulating Robert Robinson to
draw a scheme for the mode of cyclization in the hydrocarbon-sterol conver­
sion (14). Sir Robert's discussion of some of the mechanistic problems his
scheme raised included the curious but for the times perhaps not atypical
statement: "We must not allow the biogenetic tail to wag the chemical dog."
In attempts to prove conversion with radioactive biosynthetic squalene, I
spent a fmstrating but otherwise most enjoyable summer at the Biological
Station in Bermuda. All I was able to learn was that sharks of manageable
length are very difficult to catch and their oily livers impossible to slice. Much
labeled ao;:tate was wasted in these experiments. Fortunately, Robert Lang­
don, working on his PhD thesis in Chicago, used the simpler, more sensible
approach of feeding radioactive acetate to rats along with unlabeled squalene,
experiments that proved the squalene-cholesterol conversion.

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In 1952 I presented our latest results at an organic colloquium in the
Harvard chemistry department, commenting on Robinson's hypothetical
scheme for the cyclization of squalene. If my memory is correct, I made the
point that the 30-carbon-containing lanosterol would be an attractive first
cyclization product but for the fact that the site of the side chain attachment to
steroid ring D, whether at CIS or Cl7, was still unsettled. Later in the evening,
Robert Woodward asked me whether I had seen the issue of Helvetica
Chimica Acta that had just arrived. I had not and was delighted to learn that
Ruzicka and his associates had now definitive evidence for the Cl7 alterna­
tive, proving lanosterol to be a 4,4' ,14 trimethyl derivative of cholesterol.
Woodward and I proceeded to draw a cyclization scheme (8) with the object
of accommodating the lanosterol structure as an intermediate on the way to
cholesterol. That some methyl groups had to be shifted around in the process
did not disturb us. The idea was testable since the predicted but only partly
established distribution pattern of acetate-methyl and acetate-carboxyl in
labeled cholesterol would distinguish between Robinson's scheme (A) and
ours (8). Specifically, the arrangement of 4 of the 27 acetate carbons in
cholesterol would be altered. On my return to Chicago I proceeded to degrade
an appropriately labeled sample. The origin of Cl3, one of the critical carbon
atoms, conformed with scheme 8 but not A (15). One year later, during my
sabbatical stay in Ruzicka's Institute for Organische Chemie at the ETH in
Zurich in 1953, I was able to add to the evidence by establishing the origin of
another of the four critical carbon atoms (C7). This was the last time all of the
experimental work was my own. I had decided to work with my own hands in
spite of Ruzicka's skepticism. On arrival I had told him my research plans
whereupon he asked me when I had received my PhD. To my reply that it was
fifteen years ago, he responded, "Then you no longer know how to do
experiments." I refused to be deterred, and when six months later I could tell
Vlado Prelog that I had verified the prediction, his comment, with a twinkle in
his eyes, was "Oh, how dull!" This damper reminded me of Enrico Fermi's
statement: "Experimental confirmation of a prediction is merely a measure­
ment. An experiment disproving a prediction is a discovery." I do not
necessarily share this view.

The biggest remaining gap still to be filled in the biosynthetic pathway
concerned the steps intervening between acetate and squalene. It was by
chance that the key intermediate mevalonic acid was encountered in the
course of studies on bacterial growth factors. This discovery (Folkers et al
1956) paved the way for the identification of the squalene precursor
isopentenyl-pyrophosphate, the long-sought "biological isoprene unit" ( 16,
17). An account of these developments and the essential contributions of other
laboratories (Cornforth & Popjak, Lynen) is given elsewhere ( 11). Another 20
years passed before I left the intriguing and still unfinished problem of sterol
biosynthesis.

SUMMING UP 15

Annu. Rev. Biochem. 1987.56:1-18. Downloaded from www.annualreviews.org In 1954 our family moved east with anticipation that was tempered by
Access provided by 50.116.19.84 on 09/04/16. For personal use only. regrets to part with friends and colleagues. Ever since I had arrived in
Chicago, Earl Evans had given me all possible encouragement. He had
become a close family friend. We had no reason to leave except for the
yearning to live and bring up our children in a less urban environment. Going
to Harvard meant one major change among others. As a member of the
chemistry department I was expected to conform with the prevailing teaching
requiremell1ts. My teaching load, now 3-4 times what it had been in Chicago,
seemed inordinately heavy, but in retrospect I have no regrets. Teaching an
entire undergraduate course forces one to read outside one's field of
s pecialization.

By the 1950s, microbes and microbial mutants had become popular and
proven in many respects superior to rat liver as systems for studying
biosynthetic pathways that are often though not always shared by animals and
microorganisms. The elegant elucidation of aromatic biosynthesis already
mentioned was an outstanding example of the power of the mutant technique.
In order to ease the effort of adjusting from familiar to unfamiliar biological
systems, I enrolled in the famed microbiology summer course taught by C. B.
van Niel at the Hopkins Marine station in Pacific Grove. To qualify for the
course, one's record had to be satisfactory in the sense that one was not
tainted by prior educational exposure to microbiology elsewhere. The ex­
ceedingly demanding course, a bravura performance and a model of pedago­
gy, taught me important lessons that were to influence much of my later
research. First of all, the student was made aware of the enormous variety of
microorganisms and their diverse life-styles. Second, I learned from van Niel
that Nature allows the investigator to choose organisms uniquely suited for
studying a specific biological phenomenon. The awareness that such choices
exist is a valuable asset perhaps no longer emphasized sufficiently in the
teaching of biology. I benefited especially from one piece of information van
Niel mentioned casually during one of his lectures. The class was told that
Saccharomyces, the organism that led to Pasteur's discovery of "la vie sans
air, " is in fact microaerophilic, not a strict anaerobe. This bit of information
came from a paper by Andreasen & Stier ( 18), who had noted that in the strict
absence of oxygen yeast fails to grow unless supplied with a sterol and an
unsaturated fatty acid. I was pleased I could offer to the class a partial
biochemical rationale since our laboratory had just proven the origin of the
sterol hydroxyl group from molecular oxygen (19). In experiments stimulated
by the proposals of Ruzicka (20) and Eschenmoser et al (21) that an
electrophillic species of oxygen (OH+) initiates the cyclization of squalene to
sterol, T. T. Tchen was able to show that the OH group of cholesterol is
indeed derived from O2 not from water. It was the same experiment
Schoenheimer had asked me to do 15 years earlier. T. T. Tchen had traveled
to New York and returned with a glass balloon containing what was probably

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the world's total supply of 1802 gas. David Rittenberg had generously donated
it. The positive result had important ramifications, stimulating my interest in
oxygen as an essential biosynthetic reagent, and led me to investigate some
consequences for aerobic versus anaerobic patterns of metabolism. To exam­
ine the oxygen requirement for the synthesis of oleic acid, implied by the
nutritional requirement of anaerobic yeast, became D. Bloomfield's research
project after I had returned east. Working with yeast microsomes he demon­
strated that oxygen was indeed essential for the stearoyl-CoA�oleoyl-CoA
conversion (22). It was an obvious question to ask next, obvious at least to
someone indoctrinated by van Niet, how unsaturated fatty acids arise in
obligate anaerobes as they do. Possible choices were (a) an electron acceptor
other than O2, or (b) an entirely different mechanism. Guided by studies on
the structures of olefinic acids in lactobacilli (K. Hofman), we could eventual­
ly formulate and document a bacterial mechanism for olefin formation, novel
in that it involved dehydration of medium-chain �-hydroxy acids rather than
oxidative dehydrogenation of the corresponding saturated acid (23). The
requisite enzyme, �-hydroxydecanoyl thioester dehydrase, that we isolated
from E. coli, catalyzed the postulated olefin-generating dehydration step.
Fortunately the enzyme also functioned as a reversible {3,�a,{3 CIO-enoyl
thioester isomerase. Conveniently for enzyme assay but also of major conse­
quence later on, a peak at 263 nm appears in the �,y-a,� enoate isomeriza­
tion. In crude E. coli extracts these transformations required the presence of a
heat-stable protein (24), with properties reminiscent of a similar factor Vage­
los and coworkers had just shown to be essential for fatty acid synthesis in Cl.
Kluyveri (25). Before proceeding further with our studies Roy Vagelos and I
had arranged to get together during the 1961 federation meeting in Atlantic
City. Over cocktails, Roy and his coworkers Al Alberts and Peter Goldman,
and Ann Norris, Bill Lennarz, and I from my laboratory, discussed the
possible role of the heat-stable protein, known since as ACP, or acyl carrier
protein (R. Vagelos, S. Wakil). Our two groups readily agreed not to compete
but to pursue their separate objectives that had led to the chance discovery of
ACP. Roy's laboratory was primarily interested in the mechanism of chain
elongation, while we wanted to know how unsaturated fatty acids are formed
anaerobically. I believe neither side had reasons later on to regret this
amicable arrangement.

The substrate for dehydrase was unavailable commercially, a fortunate
circumstance as it turned out. We prepared it by Raney-Ni reduction of
3-decynoic acid to the corresponding 3-0lefin and subsequent thioesterifica­
tion with N-acetylcysteamine (NAC). One of several identically prepared
batches of 3-decenoyl NAC, luckily not the one made first, totally failed in
the optical test with dehydrase. David Brock's detective work provided the
first clue. In the run in question Raney-Ni reduction was incomplete, leaving

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5% of the acetylenic decynoic acid unchanged. Subsequent thioesterification
therefore afforded a mixture containing 95% olefinic thioester along with 5%
3-decynoyl-NAC. Was this contaminant to blame for the failed assay? The
answer se�:med to be yes since addition of this impure substrate to a dehydrase
assay system completely inhibited the isomerization of authentic 3-decenoyl
NAC. When pure 3-decynoyl thioester was prepared and tested, it inhibited
dehydrase noncompetitively and irreversibly with a K, of 1 x 10-7 M (26).
Complete enzyme inactivation occurred with 1 mol of inhibitor per mole of
enzyme and led to covalent modification of a single histidine residue at the
active site (27). To be effec�ive, the inhibitor had to be structurally identical
with the substrate in all respects except for substitution of the olefinic bond by
a triple bond. During one of the daily afternoon coffee hours, a cherished
tradition I inherited from my days in Schoenheimer's lab and have continued
ever since, the chemistry of the acetylene thioester--enzyme histidine interac­
tion was the subject of lively discussion. It produced the important bit of
information that acetylenes readily isomerize to the corresponding allenes.
Intriguingly, George Helmkamp had already seen a gradual spectral change
when decynoyl-NAC was kept in ethanolic solution. A new peak absorbing at
263 nm appeared and increased with time. Evidently the acetylenic thioester
isomerized spontaneously in a reaction analogous mechanistically to the
normal {3, y-a,{3 enoate transformation that dehydrase catalyzes. To our sur­
prise the i.nhibitory potency of the solution increased rather than declined.
Indeed, the pure allene, 2,3 decadienoyl-NAC, inhibited dehydrase much
more rapidly than the acetylenie isomer, proving that the allene was the true
inhibitory species (28). Still to be answered was the question of whether the
spontaneoills acetylene-allene transformation was sufficiently rapid to account
for the result or whether the isomerization was enzyme-catalyzed. This issue
was resolved by comparing kinetic isotope effects during dehydrase inhibition
by 2-dideuterio 3-decynoyl-NAC and 2,3 deuterio decadienoyl-NAC. A kHi
ko of 2.3 was found for the acetylenic thioester, while it was unity for the
allenic analogue. Enzyme-catalyzed removal of the proton from the acetylenic
thioester proved to be the rate-limiting step in the interaction between in­
hibitor and enzyme. Since the same enzyme that performs the physiological
enoyl thioester isomerization during unsaturated fatty acid synthesis in E. coli
also catalyzes the acetylene-allene isomerization, we were dealing with the
rather unique case of an enzyme promoting its own destruction; i.e. the
catalyzed transformation of a substrate analogue generated an active site
probe of extreme chemical reactivity. It is fortunate that in this instance, as
in many others, enzyme specificity is a matter of degree, not absolute. Today
the phenomenon is more popularly, but perhaps not quite aptly, known as
"enzyme suicide." I have argued, not very successfully, that this an­
thropomorphical term connotes a deliberate act on the part of the affected

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enzyme, but this is not strictly the case. Dehydrase falls prey to trickery
because it fails to distinguish between the C", protons of the acetylenic and the
olefinic substrates, an error that seals its fate . "Mechanism-based enzyme
inactivation" is gaining ground for describing the phenomenon , but whether it
will catch on remains to be seen. I have always been quite indifferent on
matters of terminology as long as the meaning is clear.

At least in principle, 3-decynoyl-NAC comes one step closer to Paul
Ehrlich's concept of a "magic bullet . " The substance inhibits the growth of E.

coli and various other bacteria (29), an effect that is fully relieved by

supplying the organism with oleic or vaccenic acids, the ultimate products of
the inhibited reaction. The inhibitor seeks out a single susceptible enzyme.
Animal cells that generate oleic acid by an entirely different mechanism lack a
comparable target and therefore 3-decynoyl NAC is nontoxic to animals.
Unfortunately the compound failed the most critical test a potential antibiotic
must pass, i.e. to protect an organism against infection. The reasons are not
known, but conceivably the lifetime of the thioester is too brief. Metabolic
instability or side effects cannot be anticipated , and therefore the hopes for
rationally designed therapeutic agents must remain guarded.

The subject of anaerobic versus aerobic life-styles in the context of evolu­
tion has continued to hold my interest ever since it came to my attention in van
Niels' course . An example is the essential role of oxygen in the conversion of
squalene to sterol. This requirement necessarily dates the appearance of
cholesterol and cholesterol-derived metabolites to an era that followed or
coincided with the arrival of aerobic cells during evolution. I pursued this line
of reasoning in an essay contributed to a "Festschift" for Severo Ochoa on the
occasion of his 70th birthday (30). Here I considered the structural and
functional consequences arising from the stepwise enzymatic modification of
the lanosterol molecule during conversion to cholesterol. Speculating on the
motives of nature , I postulated that along with the sequential departure of
the three nuclear methyl groups, in the order 14a-methyl, 4a-methyl, and
4f3-methyl, the fitness of the molecule improves, reaching perfection with
cholesterol . If this could be documented, functional improvement should
parallel the sequence of steps the contemporary pathway employs . The results
of subsequent research in recent years, mainly by Jean and Charles Dahl, both
with model membranes and sterol auxotrophs , support this notion . Fitness for
biological function, not chance , appears to be the driving force for structural
modifications of a biomolecule (3 1 ) . As for the function of sterols in mem­
branes , the chemist could not have improved on nature--or perhaps has not so
far. I am intrigued by the notion that hierarchies or Darwinian evolution are
manifest at the level of small molecules as well as at the organismic and
genomic level. In the course of these studies we have been led to a trail we are
still following. There are indications for a novel function of the sterol

SUMMING UP 1 9

Annu. Rev. Biochem. 1987.56:1-18. Downloaded from www.annualreviews.org molecule, involving control of phospholipid biosynthesis. Perhaps cholesterol
Access provided by 50.116.19.84 on 09/04/16. For personal use only. started out as a hormonelike molecule serving as signal for the assembly of
certain cell membranes . I hope to pursue this research a while longer.

One thought comes to mind once the hectic pace of teaching and research
becomes a memory. Whatever the motives , whether curiosity or ambition­
usually a combination of both-no- ly near the end does one fully appreciate
the rewards and privileges that go with a career in science . So much the better
if the results should prove to have some degree of permanence. Science is
indeed a glorious enterprise, and has been for me, I admit, glorious entertain­
ment.

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