An Interview with Manuel and Ann Varela: Paul Boyer and ATP

Mar 12, 2020 by

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1) Paul Boyer was born in the great state of Utah—but he was not a very active Mormon—when was he born and where did he go to school?

Dr. Paul Delos Boyer, a 1997 Nobel Laureate, is most famous in biochemistry circles for his mechanistic studies of the enzyme ATP synthase, which produces the energy-rich molecule ATP that is essential for fueling all cellular functions. Boyer was born on the thirty-first day of July, in 1918, in Provo, Utah, to parents Dell Delos Boyer and Grace Guyman. While the details of his elementary education are unclear, it is known that he attended high school in Provo, UT. Boyer enrolled at Brigham Young University, earning his B.S. degree in the field of chemistry in 1939. He then moved to Madison, WI, to join in the graduate school at the University of Wisconsin, where he took his Ph.D. degree in biochemistry in 1943.

Afterward, he became a faculty member at various universities at Wisconsin, Stanford, and in Minnesota before eventually settling in as a professor of biochemistry at the University of California, Los Angeles (UCLA), in 1963.

2) ATP—what exactly is it, and why is it important?

The term ATP refers to biological energy, and the acronym stands for adenosine triphosphate. In short, without the ATP molecule, there could be no life. Living beings use living energy ATP to perform their vital biological work, such as tasks involved in their metabolism during a meal, the movement of their muscles, the beating of their heart, or even to conduct their brain functions, such as in thinking or memory. The kinds of biological functions that are achieved by all living beings on Earth using the energy of ATP are profound.

The ATP molecule stores the biological power. When the living being needs its energy, the ATP molecule is then broken up by cleaving a phosphate molecule from the ATP. The phosphate cleavage produces a smaller, less energetic ADP (adenosine diphosphate). The hydrolyzing of the ATP molecules makes ADP and phosphate. The ATP breakage releases a great deal of this stored biological energy, which is then used to help accomplish the required natural tasks needed for staying alive.

ATP hydrolysis is performed every single second that the living being remains alive. In a human being, for example, it is estimated that an average adult breaks apart somewhere between 50 and 75 kilograms of the ATP each day. Thus, the ordinary human being makes and breaks apart one and a half kilograms of ATP every hour that he or she lives. This estimation corresponds to approximately 10 million molecules of ATP that are hydrolyzed per cell every second! Therefore, a living cell uses up all of its stores of ATP every 30 seconds and has to regenerate the ATP continually. The seemingly never-ending process only stops when the organism dies.

3) For several years, he was at Stanford–apparently working on some war-related efforts, which were attempting to examine or explore the stabilization of serum albumin for transfusions for the war effort. What do we know about the outcome of his work- and what exactly was he trying to accomplish?

Boyer’s work in this area began during the early 1940s during World War II. During this time, much effort had been devoted to the newly developed blood plasma transfusion program, headed by Drs. John T. Edsall and Edwin Cohn from Harvard. Their overall goal was to extend the shelf life of the life-giving blood plasma. It was important in treating wounded GIs who needed blood quickly.

Boyer and others were commissioned as part of an overall effort to use heat to denature contaminating the hepatitis virus and other infectious microbes in the blood plasma without also harming the utility of the plasma proteins themselves.

Boyer’s contribution at Stanford University involved his discovery that certain fatty acids could be used to stabilize the blood plasma protein fractions. These fatty acids contained long-chains of hydrocarbons, and Boyer found that the fractionated blood plasma preparations could withstand higher temperatures without harming the necessary serum albumin component. To Boyer’s delight, he discovered that his fatty acid supplement tended to circumvent the problem of excessive viscosity that was produced by exposure to denaturing agents like guanidine or urea. Boyer’s protective fatty acid was called caprylate, and it effectively shielded the valuable albumin protein in the urea- and guanidine-exposed blood plasma.

Many years later, in 1995, Boyer reviewed the outcome of this war effort during a 50th-anniversary celebration honoring the work. He wrote about follow-up work, which demonstrated that it took ten caprylate fatty acid molecules to protect the serum albumin effectively. Boyer also noted that the fatty acids also sought out the confounding urea and made bonds with them, as well, thus possibly shielding the albumin further from the caustic urea.

During this look back on his war work 50 years later, Boyer checked whether his fatty acid supplement was still used during blood plasma albumin fractionation. To his delight, Boyer found that after all that time, it was!

Because of this serum albumin work during the Second World War, Boyer was motivated to continue studies of protein structure and their conformations. The war experience ultimately led Boyer to study another famous protein, called ATP synthase. Thus, it proved to be a rewarding career for Boyer.

4) Boyer introduced kinetics, isotopic, and chemical methods for investigating enzyme mechanisms. First, why study enzyme mechanisms- and can you describe the kinetic, isotopic, and chemical methodologies for looking at these various enzyme mechanisms?

Boyer elucidated the molecular mechanisms involved in making the critical ATP molecule for living cells. He used three distinctive types of methods. They were based on kinetic rates of protein enzymes, isotope tracers, and chemical behaviors. Let us consider each of these methodological approaches separately.

First, regarding kinetics, it refers in this case to Boyer’s work with the so-called catalytic activities of enzymes. Here, we are referring to the ability of a protein to conduct its biochemical function, which is to bind to a dedicated substrate and chemically convert it to a new product and doing so in a most efficient manner possible.

The enzyme will conduct its biochemical process without being destroyed in the course of its chemical action. The survival of the enzyme during its chemical activity allows it to perform its chemistry process repeatedly. Thus, the enzyme molecule essentially becomes a catalyst. The catalytic behavior of an enzyme can be measured in terms of its kinetics, i.e., the rate of the chemical reaction that the protein catalyzes.

The study of enzyme kinetics involves measuring how fast a biochemical reaction occurs, i.e., chemical reaction velocities. If one measures the speed of the catalytic reaction as a function of the concentration of the substrate reactant, one observes that the rate levels off towards a plateau, gradually approaching but not quite reaching the maximum of the enzyme’s velocity.

Analysis of the initial reaction speeds that occur during enzymes dynamic behaviors also allow biochemists to tell how well a substrate interacts with the protein. This so-called affinity character between enzyme and substrate thus permit the regulation of the catalytic behavior. The concentration of the substrate [S] at which the enzyme’s initial velocity (V0) is half of the total maximum velocity (Vmax) is called the Michaelis constant, or Km. That is, this substrate concentration equals the Km when the initial catalytic rate is half of the Vmax. This regulation becomes essential in all avenues of enzymology. Importantly, Boyer’s kinetic studies allowed his elucidation of enzyme mechanics.

The second term, isotopic, refers to Boyer’s techniques involving radioactive tracers in his attempts to study enzyme catalysis of a protein known as F1 ATPase, which is a transporter of protons across the cellular or mitochondrial membranes. The F1 ATPase molecules were known to consist of five subunits, designated in Greek lettering, as α, β, γ, δ, and ε. There are three molecules each of the α- and β-subunits on a given F1-ATPase and one molecule each of the γ, δ, and ε sub-units. Thus, the full complement of these subunits is denoted as α3 β3 γ δ ε. On the same F1-ATPase molecule, there are three so-called catalytic sites, known to reside somewhere within the β-subunits.

Protons are known as hydrogen atoms with a +1 electrical charge (H+). As the proton moves across the membrane through the F1 ATPase, a molecule of ATP is broken apart, a process called ATP hydrolysis, producing an ADP molecule plus a free phosphate molecule. The phosphates consist of a single phosphorous atom (P) connected to four oxygen atoms, PO4-2, which has a net negative charge of two, -2, as two of the oxygen atoms carry the negative charges. These free-floating phosphates are also often referred to as inorganic phosphates, Pi.

During Boyer’s isotopic studies, he labeled oxygen atoms that belonged to the phosphates with an atomic derivative of the oxygen called 18O. Using these 18O isotopes of oxygen, Boyer marked the phosphates. He called these new chemicals, “18O isotopomers of Pi.” Boyer then examined the 18O-labelled free phosphates as they made contacts with the F1 ATPase proton transporters. He found that isotopic oxygen atoms on the phosphates interacted with each of the three catalytic regions, the β subunits, of the F1 ATPase proton-transporting enzyme.

Boyer thus deduced from the so-called isotopic 18O exchange studies that as the enzyme made ATP, it could not come off the protein until more of the ADP and phosphate made connections to neighboring catalytic sites on the F1 ATPase enzyme. Furthermore, Boyer showed that if ATP was present at relatively lower concentrations, then the opposite situation was at hand. That is, ATP would break apart into ADP and phosphate and not come off until another ATP made contact with another catalytic site on the enzyme.

Third, in terms of chemical methods, Boyer was involved in the chemical modification of the catalytic sites of the F1-ATPase molecule. The purpose of these experiments was aimed at blocking the β-subunits to inhibit the catalytic properties. Boyer used so-called cross-linking studies to modify the machinery of the F1-ATPase chemically. The resulting chemical modification of these β-subunits resulted in their inability to undergo conformational changes during their attempts at catalysis.

Boyer’s ingenious interpretation of these types of studies, combined with the results of the kinetics mentioned above and isotopic analyses, ultimately led Boyer to conclude that the enzyme catalysis behavior involved a rotation of the ATP synthase enzyme during ATP generation. The discovery was called rotational catalysis, and it was to earn Boyer the Nobel.

5) With John Walker, he earned the Nobel Prize for discovering “enzymatic mechanisms that underlie the biosynthesis of adenosine triphosphate (ATP).” Why was this so important a discovery?

Because ATP lies at the heart of the biological energy for all living cells, the ATP molecule is necessarily required for all life to occur. The ATP molecule is the central entity that underlies all of the components in the bioenergetics field. It may be said that ATP is the “vital force” that makes all individual cells and aggregations of multiple cells constitute living beings!

Sir Dr. John Ernest Walker, from England, elucidated the molecular structure of the ATP synthase, and Dr. Boyer discovered that its various components rotated during its catalysis functions. Boyer’s Nobel discovery was referred to as, thus, rotational catalysis. He had hypothesized that the active sites on the F1 section of the ATP synthase took turns making ATP while it turned around on its axis! In one of his seminal review articles, published in 1997 in the famous Annual Review of Biochemistry, Boyer called his ATP synthase a “splendid molecular machine!”

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The ATP synthase molecule has two essential factors, called FO and F1. The FO factor is called as such because the chemical called oligomycin was critical to its inhibition, and the F1 factor was named as such because it was the first of the elements to be purified from the mitochondria of eukaryotic cells. The FO part of the ATP synthase resides in the membrane of prokaryotes and the inner membrane of the mitochondria. The FO element functions as a proton pump. The F1 part of the ATP synthase is a knob-like structure. This F1 knob is peripherally located to the membrane and undergoes the enzyme catalysis activity.

When knowledge of Walker’s ATP synthase structure is combined with Boyer’s view of rotational catalysis, one can see clearly how the mechanism of ATP synthesis occurs. In biochemical circles, the biological device is known as the binding-change model. In this model, below, the F1 knob is viewed from the top. It is said to rotate as it catalyzes the formation of ATP. It is a remarkable molecular process that helps make life possible.

Incidentally, Boyer and Walker shared the Nobel with Dr. Jens Christian Skou, from Denmark, and who had discovered another famous ATPase, namely, the so-called sodium/potassium ATPase (Na+/K+-ATPase), a molecule which is supremely talented in muscle contraction, and brain function such as nerve conduction. The Na+/K+-ATPase is essential in maintaining the intracellular concentrations of sodium at low amounts and extracellular amounts of potassium at high levels.

6) During his illustrious career, he also worked with Professor Hugo Theorell investigating the mechanisms of alcohol dehydrogenase. Why is this important and relevant?

The alcohol dehydrogenase enzyme is harbored by a multitude of living organisms that participate in the metabolism of alcohol, including humans. The protein is located in the liver of these living beings, and as such, the ethanol is oxidized. In microorganisms, such as yeasts or bacteria, the process may be reversed as in fermentation, in which ethanol is produced in large quantities.

Professor Axel Hugo Theodor Theorell, from Sweden, earned the Nobel in 1955 for his work on the nature and effects of enzymes that catalyzed oxidation. In particular, Theorell studied cytochrome c peroxidase, catalase, and alcohol dehydrogenase. Boyer had studied briefly in the Theorell laboratory at the Nobel Medical Institute, which was housed at the Karolinska Institute. Boyer had garnered a prestigious Guggenheim fellowship to study the alcohol dehydrogenase enzyme in the Theorell lab during the same year that Theorell took the Nobel.

Together, Boyer and Theorell studied alcohol dehydrogenase enzyme extracted from the liver of test animals. The team found that the enzyme formed new bonds with its co-factor NADH (the reduced form of nicotinamide adenine dinucleotide, NAD+). As the enzyme-NADH complex formed, the co-factor possessed a higher degree of fluorescence emission. The new finding made it possible for the experimenters to measure enzyme activities readily as well as more accurately.

The alcohol dehydrogenase enzyme catalyzes the conversion of the acetaldehyde to ethanol. As the enzyme works, it converts NADH into H+ to NAD+. Boyer and Theorell took advantage of the new technological improvement in enzyme activity measurements to observe the kinetics of NADH dissociation from the alcohol dehydrogenase under different pH levels. Boyer and Theorell further exploited that ease of enzyme activity measurements to use ethanol to out-compete the NAD+ for binding to the enzyme, thus, gaining new knowledge of a precise mechanism for determination of the dissociation constant between a complex formed by NAD+ and the alcohol dehydrogenase.

Their methodological discovery for enzyme activity measurement allowed Boyer and Theorell to further deduce the dissociation constants for other enzymes, especially for those involved in fatty acid metabolism. The collaborative work resulted in the finding by Theorell’s lab that as the length of the fatty acid hydrocarbons increased, the strength of the binding for the oily substrate to the enzyme was enhanced.

7) Providing energy to cells- in a sense, may summarize his work—am I correct in this assumption?

You are correct. The energy in question is the ATP. Boyer managed to elucidate the biochemical mechanism by which ATP synthase formed the cellular energy. The mechanism is known as rotational catalysis because the ATP-forming part rotates like a wheel in the membrane! This groundbreaking discovery by Boyer forms the basis of the Nobel Prize bestowment to him.

As the enzyme turns around, ADP and phosphate come together, forming new chemical bonds and forming ATP in the meantime. The rotational catalysis mechanism ensures that the ATP making process occurs efficiently. The ATP energy thus created can then be used for a variety of purposes in the cell.

ATP is used to energize a living cell so that it can undergo its activities. Some of these processes use the ATP molecule to undergo anabolism, in which specific new molecules that a cell needs are built. The building process is known as biosynthesis.

The biosynthetic system of a cell can use ATP to make new proteins that enable a cell to specialize its functions. Further, ATP can be used to build new membranes by synthesizing fatty acids and glycerol backbone precursors. A living cell can also use ATP to synthesize DNA to specify the code of life. Cells can make new sugars and assemble them to form more substantial complex carbohydrates, for energy storage and later retrieval.

Tissues can also exploit the energy stored in ATP. For example, muscles utilize ATP to make contractions to mediate the physical movement of a limb or even of an entire living organism, like a human or any other animal. Furthermore, cardiac muscle uses ATP energy to permit a beating heart to pump oxygen laden blood to each of the body’s cells and tissues. As such, ATP, whether in skeletal or cardiac muscle, converts the energy in its hydrolysis into mechanical work.

ATP is also used by all living organisms to simply acquire needed biological molecules for life, such as water, ions, or nutrients. Specific proteins called transporters are embedded in the cellular membranes of all living organisms on Earth. These transport proteins can actively translocate their dedicated substrates across the biological membrane to the other side. During these membrane-associated transporter functions, ATP can be hydrolyzed to accumulate solutes on one side of the membrane. Thus, cells can use ATP to concentrate needed biomolecules, or they can use ATP to keep growth inhibitory substances from cell entry.

ATP makes each of these critical biological processes possible. The ATP molecule is necessary for all forms of life on Earth, from microbes to humans. Therefore, ATP is considered by biochemists to be the world’s universal energy currency for life. Without ATP, there can be no Earthly life.

8) Boyer’s work was described as “the study of enzymes, particularly to the study of oxidative phosphorylation.” Why is this realm important, and what exactly is oxidative phosphorylation?

The ATP energy of the cell permits the cell to continue living. One important issue arises, however, from the fact that ATP is the so-called universal currency of energy: namely, what is the power used to make the ATP in the first place? That is, how does one make ATP if there is no ATP to make it? If a cell is ATP depleted, then how does it begin to make more of it, if there is none to use?

The question is reminiscent of another famous one—which came first: the chicken or the egg. In biological terms, which comes first: the ATP or the energy to make it? However, if ATP is the energy, and it has gone, then where is the power to make it going to come from if there is not any power already present?

The solution to this putative quagmire is the oxidative phosphorylation you asked about above. Oxidative phosphorylation solves this seemingly impossible imbroglio. Sometimes referred to simply as “Ox-Phos” the oxidative phosphorylation system is a means of providing energy to make ATP.

As the cell acquires nutrients, it begins the metabolic process of catabolism to break down these nutrients into smaller metabolites. As it does so, some ATP molecules are formed during certain biochemical reactions along the way. Specific metabolic pathways have been developed in the course of the evolution of life and involve glycolysis and the Krebs cycle.

Glycolysis involves the oxidative breakdown of sugars and other nutrients, such as proteins and fats. The glycolytic pathway has been known by additional terms, such as the Embden-Meyerhoff-Parnas (EMP) pathway or simply the Embden-Meyerhof (EM) pathway. Likewise, the Krebs cycle is also known as the tricarboxylic acid cycle (TCA) and the citric acid cycle (CAC). However, during some of these glycolytic and Krebs cycle biochemical reactions, electrons are removed from the intermediates, a process known as oxidation (electron loss).

The NADH co-factor is also an electron carrier. So is FADH2, which is short for flavin adenine dinucleotide. Together, NADH and FADH2 constitute universal electron carriers, and they are two players in the overall oxidative phosphorylation.

The electrons from our oxidized foods are gathered to the respiratory chain system of cells. The respiratory chain has also been called the electron transport chain. In any case, while electrons are carried by NADH or FADH2 to the chain (from the broken down nutrients), the chain receives the extracted electrons. Some of the respiratory chain’s components send outward many protons, whether in the prokaryotic membrane or the inner membrane of the mitochondrion of eukaryotes. All living cells carry out the proton transport by their respiratory chains.

Importantly, the protons will pile up on just one side of these membranes, but not on the other side! The proton concentration on one side will stay relatively high while, on the other side, the proton amounts remain relatively low. This difference in proton concentration across the membrane is considered by biochemists to be another form of energy! This energy is used to make ATP. The phosphorylation aspect of the oxidative phosphorylation process is the attachment of a phosphate to ADP to result in the production of the ATP.

This second type of energy takes the form of a tremendous difference in the amounts of protons on one side of the biological membrane versus those on the other side. The proton concentration difference is called a proton gradient. Sometimes, the gradient involves sodium; thus, the difference is called an ion gradient, instead. Whatever the case, the ion gradient phenomenon is energy—it is this energy usage results in the synthesis of ATP.

The ion gradient energy has been referred to by many other names. For instance, it may be called the electrochemical gradient of ions (or protons or sodium), or it may be called the proton-, or sodium-, or ion-motive force.

The method in which the ion-motive force is used to make ATP works through the ATP synthase enzyme. The ion, whether proton or sodium, will bind to the FO channel and get transported through it across the membrane. Next, as the FO sub-unit rotates, according to Boyer, the F1 sub-unit will catalyze the phosphorylation of ADP, making ATP. Thus, ATP production is the result of the oxidative phosphorylation process.

Without the Ox-Phos, the ATP would not be made, and the cell would die. Specific poisons, like cyanide, will prevent the oxidative behavior of the electron transport system, resulting in the dissipation of the ion-motive force and, thus, the inability to undergo phosphorylation to produce ATP. The final electron acceptor can be oxygen, an inorganic molecule, or an organic molecule, depending on the organism.

9) He died at the age of 99—two months shy of his 100th birthday—what can one say about his 99 years of contributing to science?

Interestingly, Boyer’s longevity in the scientific field involved his interaction with other scientific notables, such as professor Peter Mitchell, who formulated the so-called chemiosmotic theory of oxidative phosphorylation. Mitchell had held that the proto-motive force, generated during respiration, was the energy to drive Boyer’s ATP synthase into action.

Other aspects of Boyer’s life played prominent roles into his entry into the science of biochemistry. The death of Boyer’s mother from the terrible effects of Addison’s disease proved to be a tremendous influence on young Boyer. It had been this incident that compelled him to enter studies in biochemistry.

As a child, one of Boyer’s least enjoyable memories involved waking up at odd hours of the night to water the family garden as assigned by his parent’s irrigation schedule. In later years, Boyer attributed his acumen in biochemistry directly to the teachings of his parents. He wrote that his parents had instilled upon him the importance of honesty, logical reasoning, realistic discipline, as well as empathy and the love of others.

In 2018, on the second day of June, Boyer passed away in Los Angeles, California. I surmise that given the immense wealth of biochemical knowledge Boyer has bestowed upon the scientific world, one quote attributed to him might suffice. He had said that if societal support in favor of basic research into how living organisms function, he felt that his grandchildren would eventually be spared from the suffering inherent in losing a loved one to cancer. Boyers contribution to energy transduction of living cells also informs the relevance to cancer because tumors also have to acquire ATP to wreak their havoc. Boyer’s work advanced the cell energetics field of study tremendously. Consequently, continued scientific investigation into how one can effectively de-energize cancer cells to kill them without concomitantly affecting healthy non-cancerous cells, will most certainly help Boyer’s aspiration about the lessening of human suffering to come to a warmly welcomed fruition.

For further study and to learn more about this impressive scientist, go to:;

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