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Manuel Varela— He Shared the Nobel Prize with Krebs!

Jun 30, 2017 by

An Interview with Manuel Varela— He Shared the Nobel Prize with Krebs!

Michael F. Shaughnessy –

1) Professor Varela, Fritz Albert Lipmann is another famous biochemist- and he apparently shared the Nobel Prize with Hans Krebs.   What do we know about Lipmann and his early life and education?

On the 12th of June, just prior to the start of the 20th century, in the year 1899, Fritz Lipmann was born in Königsberg, which was at the time the capital city of the small country of East Prussia, later becoming part of Germany and which is now located in present day Kaliningrad, in Russia. Lipmann’s mother was Gertrud Lachmanski Lipmann, and his father, Leopold Lipmann, was a lawyer. Lipmann attended elementary and high schools (called gymnasiums at the time) in Königsberg, where he studied Greek and Latin.

Main influences in Lipmann’s early childhood include his older sibling by two years, Heinz; an uncle, a pediatrician who died young due to a burst appendix; and  his father, who confessed to young Fritz that he (Leopold) was not a good enough crook to make an excellent attorney. Speaking of confessions, Lipmann reveals in a memoir that he was not a very good student in gymnasium or in university.

After graduation from gymnasium, Lipmann began the study of medicine in 1917, but shortly thereafter his studies were interrupted when he was called into the Army where he conducted his medical service. At that time, the Great War (World War I) had just begun. During this time, he gained experiences in military medicine during battles and observed the Great Influenza pandemic, killing an estimated 30 million people.

With the Great War over in 1919, Lipmann and Heinz then relocated to the University of Munich, Germany, where Fritz had signed up for one academic semester, studying medicine. Lipmann then moved to the University of Berlin to finish his medical degree, in 1924. Upon the advice of his parents, Lipmann spent several months working with cadavers and microscopes, in order to better prepare him for medical studies.

However, doubts about practicing medicine began to creep in; he felt somewhat uncomfortable charging people money for keeping them healthy in return. Lastly, Lipmann took a course in Biochemistry, the study of the chemical reactions in living systems—his lecturer (professor) was Dr. Peter Rona. These experiences led Lipmann to pursue the study of biochemistry.

In his last year of medical studies, Lipmann entered Rona’s research laboratory, housed at the University of Berlin. Prof. Rona was an eminent investigator; he had collaborated with another famous biochemist, Leonor Michaelis (enzyme kinetics), and Rona had taught future Nobel Laureates Ernst Chain (penicillin production), and Hans Krebs (intermediary metabolism). Anyhow, it is in Rona’s laboratory that Lipmann published his first paper in a scientific journal, in 1924, presenting his results on colloidal chemistry. This work was also to be used for a thesis project, which was required for his medical education. He graduated in Berlin with his M.D. degree in 1924.

In Rona’s laboratory, Lipmann also published his results from his experiments working with rabbits in which he had injected with various carbohydrates, such as glucose, starch and glycogen and measured their blood glucose levels, in 1924 and 1925.

With his M.D. degree in hand, Lipmann was still greatly interested in biochemistry and research. Thus, in order to continue with this new interest in biochemistry, Lipmann felt he needed to learn basic chemistry first. Therefore, he had taken a post-graduate fellowship at the University of Amsterdam, in the pharmacology department, where he learned about the main concepts pertaining to biochemistry, in the laboratory of Dr. Ernst Laqueur. Next, he moved back home with his parents and studied chemistry for three years under the supervision of Dr. Han Meerwein, who was a prominent organic chemist at the University of Königsberg.

Dr. Lipmann then made the unprecedented move of entering graduate school for yet another advanced degree, this time the Ph.D. degree. In 1927, he was accepted with no pay at the Kaiser-Wilhelm Institute for Biology in Berlin, directed by the great scientist and biochemist of the day, Prof. Otto Warburg. At this Institute in Berlin, graduate student Dr. Lipmann entered the research laboratory of yet another famous biochemist of the time, namely, the lab of the great Prof. Otto Meyerhof, who had been a protégé of Prof. Warburg. Meyerhof had already received the Nobel Prize in 1922. Warburg was to receive the Nobel later, in 1931.

In the Meyerhof laboratory in 1927, Dr. Lipmann first conducted experiments dealing with measuring the effects upon rabbit blood sugar of yeast extract (autolysate) that was hypothesized to harbor an elusive “glucose activator,” which they had hoped would be an enzyme, called hexokinase, contained within the autolysate. Unfortunately, there was no effect on blood sugar levels.

The second project conducted by graduate student Lipmann in the Meyerhof lab involved determining whether the muscle contraction-inducing substance called acetyl choline caused the breakdown of creatine phosphate (denoted as C~P and which is used up to provide energy during acute vigorous exercise) in frog muscle. Unfortunately, acetyl choline had no effect on C~P hydrolysis, although Lipmann did manage to show that C~P was indeed split in half to form individual creatine and phosphate during the contraction of frog muscle. Incidentally, it was Lipmann himself who was the very first to invent the “~” symbol, called the “squiggle” to denote as high energy phosphate bonds. Breaking these energy-rich bonds releases the energy stored in them.

The third and last graduate project performed by Lipmann in Meyerhof’s lab entailed the examination of fluoride upon sugar breakdown, a process known as glycolysis. Lipmann demonstrated that fluoride inhibited glycolysis, an important finding. He also showed that the fluoride combined with a form of hemoglobin called met-hemoglobin to form fluoro-met-hemoglobin.

Each of the three projects were published as three separate papers in scientific journals and served as a basis for Lipmann’s graduate doctoral thesis, which he earned in 1929. At this time, Lipmann now had both an M.D. and a Ph.D., both of which firmly established his entry into the important field of biochemistry and its sub-field, metabolism.

Lipmann then entered the laboratory of Albert Fischer at the same institution in 1930 studying the growth of cells and using manometry to measure oxygen uptake into the grown cells. But the early 1930s were precarious years as Hitler’s rise to power became established. Fortunately, Lipmann and his new wife Freda Hall, an American, were able to leave Germany to accompany Fischer as he moved to his new post in Copenhagen, Denmark.

While Fischer’s new laboratory was still being constructed Lipmann travelled to the U.S. for the first time and worked briefly with Phoebus Levene at the Rockefeller Institute for Medical Research, in NY.  Working with Fischer, Lipmann studied the “Pasteur Effect,” a phenomenon in which facultatively anaerobic cells turn off ethanol fermentation when the cells are grown in the presence of oxygen. It is in Copenhagen where Lipmann converted from the study of animals to the study of bacteria.

Lipmann spent the summer of 1931 at the famous Marine Biological Laboratory (MBL) in Woods Hole, MA, where he shared a laboratory with Leonor Michaelis, another famous biochemist who studied enzyme kinetics. Many biochemists and later physiologists and cell biologists would spend each summer at the MBL in Woods Hole conducting experiments and holding seminars daily.

During the mid- and late-1930s, the situation with Hitler’s fascism grew worse, and now Denmark was deemed unsafe, as well. Fortuitously, Lipmann’s work on the Pasteur Effect got the attention of Dr. Vincent du Vigneaud at New York’s Cornell Medical College in the U.S. In 1939, Dr. du Vigneaud offered Lipmann a temporary post at Cornell, and Lipmann eagerly accepted.

In 1941, Lipmann moved Boston, MA to work as a research associate at the prestigious Massachusetts General Hospital, affiliated with Harvard Medical School.  It is here at Mass General where Lipmann conducted his famous Nobel Prize worthy work with Acetyl-CoA, a central metabolite. At Harvard Medical School, Lipmann moved through the academic ranks, first as a research fellow and finally as a full-professor in the biological chemistry department.

Lipmann stayed at Harvard Med till 1957, when he moved to Rockefeller and eventually became professor emeritus.  His later works concerned studies of the metabolite carbamyl phosphate and its relationship to the biosynthesis of another metabolite called citrulline, plus works on sulfur biochemistry, and later studies pertaining to protein synthesis, or translation. He passed away on the 24th of July in 1986, at the age of 87.

2) Professor Varela, we know that there are all kinds of biochemical reactions going on in the human body- we have oxygen going in, we may have just had lunch or a nap- Can you tell us about some of the MAIN biochemical reactions going on as we sit here and type and speak?

Well, after just having finished a meal consisting of food substances, like let’s say, carbohydrates, protein, some fats, and nucleic acids, an interesting process takes place as these substances are broken down in order to make energy. It may be somewhat of a surprise to you that just about everyone’s favorite food consists of electrons—at least they could be our favorites, being that all living beings, from bacteria to humans, have devoted a great deal of our biological metabolic machinery to the acquisition of electrons.

First, the electrons that surround the atoms in the meal are taken away from the food breakdown products, a process called oxidation (electron loss) or oxidative degradation, also called simply catabolism.  Next, the electrons taken from food are then brought to the oxygens that you had mentioned were going in. Along the way, the electrons that had been extracted from the molecules making up your meal are shuttled along a series of specialized proteins, collectively called a respiratory chain or an electron transport chain, embedded in the inner membrane of a eukaryotic cell’s mitochondria (an energy powerhouse) or in the membrane of bacteria.

As the electrons are shuttled along the respiratory chain, a bunch of charged hydrogen atoms (i.e., H+) called protons are pushed out through the chain, building up on one side of the membrane and forming a so-called proton gradient. This proton gradient itself is a biological energy form that’s used to make another biological energy form, called ATP (adenosine triphosphate). Overall, this metabolic system is called oxidative phosphorylation. The oxidative part is the electron removal from food breakdown products and specialized electron carriers called NADH and FADH2; whereas the phosphorylation part is the addition of phosphate to ADP to make the ATP.

The catabolism of foodstuffs involves a series of biochemical pathways, depending on which food has been consumed by the living being. The breakdown of carbohydrates, like sugars called sucrose, fructose, glucose, etc., involves a metabolic pathway, most often called glycolysis (“glyco” means sugar, and “lysis” means breakdown).  Sometimes glycolysis is also called the Embden-Meyerhof-Parnas (EMP) pathway, or even the more obscure designation, the fructose bisphosphate aldolase pathway. My favorite is the EMP pathway, because it is named after three key investigators, Gustav Embden, Otto Meyerhof, and Jacob Parnas, who participated in figuring out the centrally important metabolic pathway.

Interestingly, all living beings on Earth, from bacteria to humans, undergo glycolysis in much the same way. It is a process that was invented by our common ancestors and is unquestionably of major importance today for life.

The breakdown of fats occurs by first breaking them in half, forming a 3-carbon glycerol backbone and, depending of the type of fat, a group of fatty acids. The fatty acids are long chains of carbon hooked up to a bunch of hydrogen atoms. The glycerol is taken to glycolysis and finished up there while the fatty acids are taken apart two carbons at a time, in a process called β-oxidation, to form an extremely important metabolic intermediate, called acetyl-Coenzyme A (Ac-CoA), which then goes to the Krebs cycle.

The breakdown of protein involves the clipping off of the amino acids that make up the protein chains. The amino acids are then further taken to glycolysis and Krebs cycle. Likewise, the breakdown of nucleic acids also involves their movement to glycolysis and Krebs cycle.

Overall, the process of breaking down these food materials in the presence of oxygen is called aerobic respiration. In certain living beings, like bacteria for instance, some of which also undergo aerobic metabolism, they sometimes undergo catabolism without oxygen, a process called anaerobic respiration. Living beings that do their metabolism with or without oxygen are called facultative anaerobes. Besides respiration (whether aerobic or anaerobic), certain life forms undergo a process called fermentation, which involves glycolysis and then any one of a large repertoire of fermentative pathways.

The other half of metabolism, catabolism being the first half, is called anabolism. Here in anabolism, the energy store in the ATP molecule is used to build larger molecules, some being called macromolecules, all of which are critically useful in order for life to occur. One central theme in these metabolic systems is that they use enzymes, the substrates that the enzymes do their chemistry upon, and products that result from the biochemical processes carried out by these enzymes. In fact, several of the key biochemical steps in glycolysis are in play and are shared during some anabolic reactions, but are run in reverse directions, even sometimes using the same enzymes.

3) What can go wrong with these reactions? Obviously, if we do not get enough oxygen, the heart and brain start to die (and we start to die also).

Clearly, you have identified a key biological requirement for many living beings, namely, the necessity for oxygen. When living beings metabolize their meals, like I’ve mentioned above, the electrons that are taken away from the metabolic intermediates are frequently sent to oxygen.  In this sense, the final electron acceptor, if you will, is oxygen.

If one does not get enough oxygen in organisms that are obligate or strict aerobes, the cells, tissues, organs, like the heart or the brain, etc. will die, and consequently the organism will die, as well.  In these aerobe organisms, other forms of oxygen, such as carbon dioxide (CO2) or carbon monoxide (CO), are not good enough to serve as the final electron acceptors—in these cases, only oxygen (and in the form O2) will suffice.

Another way in which these reactions can go wrong, is when there is, e.g., a lack or deficiency of a substrate, or a missing or malfunctioning enzyme that has been produced in a living being. In the first case, substrate deficiency, it may be the result of a poor diet; and in the case of a dysfunctional enzyme, it may be due to a genetic disease in which the gene that encodes the enzyme has been altered by an inheritable mutation. Perhaps another gene encoding the synthesis of a needed substrate for another enzyme is defective, causing a loss of the needed product.

In some cases, missing or non-functional enzymes mean that a substrate builds up to abnormal levels, causing illness.  An example is glucose-galactose malabsorption, in which key transporter proteins that allow sugar entry into cells are lacking, or in the case of lactose intolerance where key enzymes in the metabolic pathway that catabolize lactose sugar is present in low levels. These metabolic conditions may require careful diet or replacement therapies.

4) Now, glycolysis—what is it and how does it fit into chemical reactions?

Glycolysis involves the oxidative breakdown of sugar, like glucose molecules, to produce the metabolite called pyruvate. The glycolytic pathway consists of a series of substrates, enzymes, and products. Along the way, electrons are collected from the intermediate metabolites and concentrated by electron-capturing molecules called NADH and FADH2. In the beginning of the pathway, some ATP energy is used up; but later on this energy expenditure is rewarded with a net profit of ATP, which can be used for other life-giving biosynthetic purposes. Again, the sugar is split into two parts, each part being metabolized further to make 2 molecules of the pyruvate.

An important biochemical action in glycolysis is called substrate-level phosphorylation.  Here, a given substrate that is phosphorylated, i.e., a phosphate attached to the enzymatic substrate, is utilized by a protein-based enzyme to transfer the phosphate to an ADP recipient molecule, making a new ATP molecule—the source of the phosphate for making ATP is a substrate—this was a new type of reaction at the time, and it occurs in glycolysis twice. It is interesting also that in cancer cells, this glycolytic pathway is usually working in a rapid mode, as cancer cells require lots of energy to grow.

Glycolysis is virtually a universal process for all living cells, whether they be bacterial in nature or human. It turns out that because we are carbon-based life forms, the most common source of carbon comes from sugar. Another interesting thing about glycolysis is that it is a central pathway. That is, whether the foods ingested are carbohydrates, protein, fats or nucleic acids, they will all be metabolized via glycolysis.

5) What is the relevance of glycolysis to Krebs cycle?

In a sense, glycolysis represents an incomplete metabolic system. It’s end-product, pyruvate, is not necessarily a complete oxidation—the sugars and other food products can actually be broken down even further, all the way to water and carbon dioxide. The Krebs cycle plays a key role in this complete oxidization. Incidentally, the Krebs cycle has also been called the TCA (tricarboxylic acid) and the citric acid cycles.

In the cases of some intermediary metabolites, they can go straight to the Krebs cycle. On the other hand, for other metabolites, before the Krebs cycle pathway can operate, the glycolysis pathway must first do so. It is in this latter situation that the work of Lipmann comes into play. Glycolysis and the Krebs cycle are connected by a reaction that Lipmann discovered. The Krebs cycle pathway allows the complete oxidation of the metabolites produced in glycolysis.

6) Apparently Krebs and Lipmann shared the Nobel Prize – Can you tell us the nature of their discovery or what they were recognized for?

You are correct about these two amazing scientists. In the year 1953, Fritz Lipmann and Hans Krebs were to share equally the honor of receiving the Nobel Prize in the category of Physiology or Medicine for their discoveries. Prof. Krebs elucidated and connected key biochemical reactions and literally cycled them upon each other to propose the cyclic nature of the pathway, the Krebs cycle. These were largely the contributions of Krebs.

Lipmann, on the other hand, elucidated the inner workings of what is considered to be the most important and central biochemical reaction ever discovered. In short, Lipmann’s Nobel discovery identified the entry point into the Krebs cycle and in doing so, made the hugely important connection between the glycolytic and TCA pathways.

Both of these pathways participate in the complete oxidative degradation of sugars, amino acids, fatty acids, glycerol, and nucleic acids while generating CO2 and water and retrieving their electrons.  The electrons are then taken to the respiratory chain so that the O2 can accept these electrons, generating proton (or other ion) gradients to be used as energy in order to make ATP energy.

In summary, Lipmann’s famous reaction, linking glycolysis with Krebs, can be denoted as follows:

Pyruvate + Coenzyme A + NAD+    →   Acetyl-Coenzyme A + CO2 + NADH + H+

The enzyme that catalyzes Lipmann’s important reaction is called the Pyruvate Dehydrogenase Complex (PDC). The pyruvate was generated as the end-point of glycolysis. The Coenzyme A (CoA) or “Coenzyme for Acetylation,” has parts of the B5 vitamin called pantothenic acid. The NAD+ is a carrier of electrons and is used as a vessel for the electrons that are taken away from pyruvate; that is, pyruvate oxidation occurs during the reaction. The electrons go to NAD+ along with hydrogen atoms, called hydrides, which are also taken away from pyruvate to form H+ (a charged hydrogen atom called a proton) and to make the NADH; the letter H of the NADH stands for the element hydrogen—thus, removal of the electrons (oxidation) from the substrate and accompanied by hydrogen atoms, is a process that is referred to as dehydrogenation. Hence, the enzyme gets part of its name, dehydrogenase, from this step; the hydrogens are lost from pyruvate.

Pyruvate is a 3-carbon molecule, and when a decarboxylation step (defined as a release of CO2) occurs to form the freed up CO2, the remaining 2 carbons that are still attached to each other are referred to as an acetate. In Lipmann’s reaction, CoA is attached to the 2-carbon acetate, forming Acetyl-CoA.  Some investigators call this an acetyl transfer.

Acetyl-CoA happens to be one of the most important metabolites ever made by living organisms. One reason for its relevance is because it’s a central metabolite between the two catabolic and anabolic systems that occur during metabolism. Lipmann and colleagues frequently referred to it as an active acetate.

Another reason why Acetyl-CoA is so critical is that it participates in the first and last step(s) of Krebs. Here, the enzyme is called citrate synthase, and like the enzyme says, it synthesizes citrate by combining the 2-carbon acetate with the 4 carbons of the end-point metabolite of Krebs, called oxaloacetate, forming the 6 carbon citrate and completing the cycle!

Thus, applying Lipmann’s famous reaction as an entry point into the Krebs cycle was a key finding.

7) I have been to Gamla Stan which is where they award the Nobel Prize in Stockholm, Sweden.  For a scientist- is the Nobel Prize basically the highest recognition a scientist can receive?

Indeed, the Nobel is the greatest prize that a scientist could ever hope to garner. Amongst scientists and many others in non-scientific fields of study, there is no other equivalent. The bestowment of the Nobel often signifies general and wide acceptance of the scientific finding, especially amongst a Laureate’s peers.  Often, when the stakes are high, an important scientific finding may have one or more detractors, and receipt of the Nobel often serves as a confirmation of the validity of these discoveries.

Acceptance of a scientific discovery involves a review of the findings by other experts in the field (often anonymously) prior to the publication in the scientific journal.  The quality of the hypothesis, the logical rationale for the hypothesis being tested, the experimental design, the chosen methods of statistical analyses, the repeatability of the findings, and the importance of the discovery are just some of the things that are heavily scrutinized by peer referees or reviewers, before an editor ever accepts the work for permanent publication. Receipt of the Nobel signifies not only a true confirmation and peer acceptance of their discoveries but also of their immense relevance. In short, the Nobel may be the greatest approval of all for an investigator who seeks the truth in scientific terms.

8) What have I neglected to ask about Lipmann?

In his memoirs, there is an interesting anecdote about Lipmann and his attempt to find a job. His post at Cornell not being a permanent job, he obviously needed a permanent post. His golden opportunity arrived in 1940 when Lipmann was to give a seminar about his Copenhagen work at an important scientific conference in Madison Wisconsin. It was a large symposium dedicated to intermediary metabolism. Virtually all of the major experts in the field would be there at the symposium. With all of his experimental accomplishments and discoveries published in the journals, Lipmann was already well known from the literature, and the symposium was his chance to shine in front of his colleagues. It turned out to be the worst presentation Lipmann had ever given.

First, he was not, at the time, terribly experienced in lecturing, and not having prepared properly for the seminar, he ran out of time. He had not anticipated the time constraints being held for each of the talks, and he consequently lectured on only half of the material before he was clocked out. Because of this one disastrous lecture with all or most potential hiring managers in attendance, Lipmann attributes his later difficulties in acquiring a job directly to this one poor performance.

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