Sir Hans Krebs (1981) - The Evolution of the Citric Acid Cycle and other Metabolic Pathways

Ladies and gentlemen, ever since it was recognised in the 1930s that some metabolic pathways are cyclic in their arrangement, for example the citric acid cycle, I have often asked myself why metabolic cycles have arisen over the course of evolution. I had not been able to answer this question until recently. As you know, acetate - acetic acid - is oxidised in the citric acid cycle into carbonic acid and water. And I discussed the question of why acetate is not oxidised directly in a paper in 1947. which then changes to glyoxalate , then via formaldehyde to carbonic acid bicarbonate, into carbonic acid. That looks much simpler at first glance. However, at the time in 1947, as well as afterwards up to about a year ago, I was unable to provide an answer to this question of why the citric acid cycle is favoured. I discussed this problem in March of the previous year with my colleague Jack Baldwin, professor of organic chemistry at Oxford, and in the course of the conversation, he developed a grand idea that might lead to answering this question. It was based on the theory of evolution through natural selection. This principle applies to competition between viable members of a species. And in this competition, viable members that are more capable and efficient multiply at the expense of viable members that are less so. And after a time, only the more capable and efficient viable members of the species survive. And this principle leads to the question of whether combustion of acetic acid via the citric acid cycle holds an advantage over other metabolic pathways. The biological purpose of combustion in living cells is decidedly not, as you know, the oxidation of the substrate, but instead the utilisation of the energy released for the synthesis ATP. And the most advantageous mechanism of oxidising acetic acid is hence the one that provides the most ATP. The maximum yield of ATP can be calculated. The yield is not directly connected to the oxidation of the acetic acid, but instead, as you have just heard in the lecture from Professor Lipmann, instead with the transport of hydrogen atoms via the chain of cellular respiration reactions to molecular oxygen. The requirement for ATP synthesis is therefore the dehydrogenation of the substrate. And therefore, the question arises of whether the direct oxidation of the acetic acid to glycolate, as is shown in this slide, has just as great a utility in terms of the synthesis of ATP as the citric acid cycle does. And the answer to this question is no, because it is impossible to dehydrogenate acetic acid using the coenzymes of the cellular respiration chain. The first step is not dehydrogenation - that is impossible. Dehydrogenation, that means the transport of hydrogen atoms to the coenzymes of the cellular respiration chain. The methyl group is namely unable to release two hydrogen atoms simultaneously. It is therefore impossible, precisely because no double bonds can be formed to remove two hydrogen atoms from this methyl group. This means then that dehydrogenation, in the sense of transferring hydrogen atoms, or a transfer to the coenzymes, either in this pyridine nucleotide or to a flavoprotein, is not possible. Now, methyl groups can nevertheless be oxidised, just as those of acetic acid in living cells can be, but only via molecular oxygen, in contrast to dehydrogenation. The enzyme (may I have the next slide please?) that achieves this reaction is a hydroxylase or monooxygenase - also called a multi-function oxydase. This enzyme is not located in the mitochondria where the energy transformation takes place, but instead in the microsomal portion. This enzyme, the monooxygenase, catalyses the insertion of an oxygen atom of molecular oxygen into the organic substrate. From this two-atom oxygen molecule, one goes here between the carbon and the hydrogen, as shown, CH2OH. Meanwhile, the second oxygen atom, the molecular oxygen-H2 atom, an acceptor must be present for the second oxygen atom, and this acceptor is NADPH2 in most cases, so that NADP and water result. And this is the general expression of the oxygenase reaction, where the RCH3 is a methyl group that cannot be dehydrogenated because of the properties of R, which is a radical. R can therefore be not just acetic acid, but instead methane (CH4) for example, which can be oxidised by microorganisms. It can also be a side chain of benzene rings, like toluene or xylene, and there is a whole series of methyl groups that can participate in similar reactions. Now, the oxidation of acetic acid to glycolic acid is not only therefore worthless from the standpoint of ATP synthesis, since it does not provide any ATP, but moreover, because the consumption of one molecule of NADPH2 for the reaction of two oxygen atoms, of the oxygen molecule, prevents the synthesis of 3 additional ATP molecules. As a result, the fact that this NADPH cannot be transformed into NADH and enters the cellular respiration chain, eliminates the opportunity of synthesising 3 more ATPs. The quantitative details of the squandered energy are now as follows: when acetic acid is oxidised via the citric acid cycle, the final yield is ten pyrophosphate bonds of ATP. Twelve are formed in connection with the reactions in the cycle, but two pyrophosphate bonds are necessary for the formation of acetyl coenzyme A from acetic acid. Now if the acetate were to be oxidised directly via glycolic acid, then only 5 or 6 ATP molecules fewer would be formed, because the oxygenase reaction cannot be connected with synthesis of an ATP and because one molecule of NADPH is consumed as a co-substrate of this oxygenase. If acetate were catabolised by the direct pathway, the ATP yield would therefore only be half as much in comparison to the ATP yield via the citric acid cycle. And the relationships are similar if carbohydrates and fatty acids are the sources of the acetyl coenzyme A rather than free acetate as the source, which generally is present in the diet only in small quantities. What I have therefore discussed so far explains not only why direct oxidation of acetate cannot compete with the citric acid cycle in relation to utility, it also provides an explanation of why the alternative to the "direct" catabolic pathway must be a cycle. to a second substance having a lower molecular weight, that is acetate to oxaloacetate, then this second lower-molecular-weight substrate must be regenerated if the primary metabolic process has taken place, which in the case of citric acid is the citric acid cycle, the combustion of the acetic acid. This is necessary, this regeneration of the second substance, the oxaloacetate, because precisely this second substance, the oxaloacetate, must be available again. It has to come from somewhere. Where is it supposed to come from? Cyclical regeneration is the only conceivable physiologically efficient mechanism. If this were not the case, then it would lead namely to the absurd consequence of a by-product from the acetic acid oxidation initially forming in stoichiometric proportions, which would be completely ridiculous. For example, an adult taking in 2,000 kilocalories per day would produce 800 grams of oxalic acid and in addition, would also require 800 grams of oxalic acid. That is all ridiculous, of course, and therefore a cycle is the sole rational and economical mechanism for organising certain metabolic functions. This permits us therefore to predict that effective utilisation of the acetate as an energy source requires bonding of the acetate to a further substance that is regenerated by a cycle. However, the question still remains now, why exactly the citric acid cycle and not other conceivable cycles arose over the course of evolution, and why acetate takes the form of acetyl coenzyme A in reactions? In this regard, it can be pointed out that the components of the cycle, including those of acetyl coenzyme A and the synthesis of the citrate from acetyl coenzyme A and oxaloacetate, and in addition, the transformation of citrate into oxoglutaric acid, already existed in life forms long before molecular oxygen was present in the atmosphere in significant quantities, therefore when life was still anaerobic. These are substances and metabolic pathways that were generally assume to already be present in the atmosphere. The atmosphere was, after all, originally anaerobic and oxygen was only introduced into the atmosphere through the assimilation of carbon dioxide by photosynthesis, and these reactions, about which I need not go into in detail, were already present because they led namely to the synthesis of important substances. This should not be glutaric acid, but rather glutamate, that is, glutamic acid is formed in this manner and in addition, the porphyrins, which include chlorophyll and cytochrome, are synthesised from oxoglutaric acid via succinyl coenzyme A. That means, then, that living organisms have drawn on reaction sequences during the evolution of the citric acid cycle which already existed in connection with other biochemical functions, such as the synthesis of amino acids and that of chlorophyll. Multiple use of available material is now a general principle of evolution and I would like to illustrate this through several examples. (The next slide, please.) Amino acids, as you know, are components of all proteins, but they have many other functions in addition. I would like to go into this very briefly: glycol, or glycine, is a precursor of creatine, of bile or choleric acid, and the porphyrins, glutathione, hippuric acid; serine becomes substances that occur in phospholipids; cystine is a precursor of taurine; arginine is a precursor of creatine. (The next slide, please). Some further examples: aspartic acid is a precursor of nucleic acid, alanine functions as a nitrogen transporter between tissues, etc. Tyrosine is the precursor of hormones, adrenaline or thyroxine, etc. I could also mention, for example, that the ATP not only plays a large role in energy transformation, but also functions as a precursor, a component of pyridine nucleotides and nucleic acids. And underlying this is the general principle of natural selection, that the circumstances in the environment are optimally utilised. Now the principle that a cyclical metabolic pathway is more efficient than conceivable linear pathways applies to all of the other cycles that I have analysed up to now, for example the ornithine cycle in urea synthesis, the Calvin cycle in photosynthesis, and the pentose phosphate pathway that produces the reduced coenzyme NADPH2. However, due to lack of time, I am unable to go into this further. The principle is always that the substance is unable to react directly, for example ammonia in urea synthesis. In a test tube under abnormal conditions, if you work in an alkaline environment and at high temperatures, then you can likewise get urea from ammonium carbonate. But under the conditions for which life is possible, neutral pH and normal temperature, here it is necessary for the molecule to take up another molecule. Just as acetate takes up oxalic acid, carbonic acid and ammonia take up ornithine. However, what I still would like to discuss, namely the details of other cycles, I cannot go into. But what I would indeed like to discuss still is the general application of the question To what extent are they superior to other theoretically conceivable pathways?" Such questions can be approached from the standpoint of modern knowledge of organic chemical reaction mechanisms, and based on this knowledge, we can predict which reactions are possible at all, and which ones are difficult or impossible. For example, and this concerns linear reactions now, why are fatty acids catabolised by Beta-Oxidation? Why not Alpha-Oxidation or Gamma-Oxidation or Omega-Oxidation? The answer is, again I cannot go into detail due to lack of time, that would be a long story: Beta-Oxidation produces more ATP, much more ATP. Alpha-Oxidation and Omega-Oxidation can occur under particular circumstances. But the oxidizing agent is not a pyridine nucleotide or a flavoprotein, but instead molecular oxygen, and the enzyme is a monooxygenase, because dehydrogenation by a flavoprotein or pyridine nucleotide is impossible for Alpha-Oxidation and Beta-Oxidation. We have to accept this, as I said, I cannot go into detail. This also explains why fatty acids do not react when they are free, but instead as thioesters of coenzyme A and also why fatty acids must be bound to a sulphur atom and not just any element. George Wald has already made this clear some time ago. This has to do with particular atomic properties of sulphur. Phosphate would be another possibility. That is also used biologically on occasion. There is also acetyl phosphate, as Fritz Lipmann has discovered, but that is only a precursor in the energy turnover of acetyl coenzyme A. And it is also clear, of course - chemistry teaches us this - just why free fatty acids cannot react. I now believe that applying modern knowledge of reaction mechanisms to the problems of metabolic pathways will be a fruitful area for future research and that developing this area will lead to a deeper understanding of the organisation of substances in life forms. I find it very satisfying that from now on we will be able to answer the question raised of why metabolic cycles have arisen over the course of evolution, a question that has been directed toward me quite frequently by students. In retrospect, the answer appears extraordinarily simple: based on evolution through natural selection. There are definitely circumstances in which a cyclical mechanism can be much more efficient than a non-cyclical mechanism. It involves the fittest in the fight for existence, survival of the fittest. As you may know, this expression 'survival of the fittest' has been criticised as a tautology. It has been said that it means nothing more than the survival of the survivor. However, this kind of criticism fails to address the arguments in relation to biochemical evolution, because, namely, the fittest can be expressed quantitatively in chemical form by the yield of ATP from combustion of organic substances, as I have discussed in the case of the citric acid cycle for example. And now, another general observation in closing: The analysis of reactions that produce energy, and one can even say the deeper study of living organisms, makes it clear that life has attained optimal organisational efficiency in the course of several billion years. This insight is very important for physicians' daily practice. Highly effective modern medicinal products, almost all of these medicines have side effects, quite disruptive and sometimes even fatal side effects, because they disturb the optimum efficiency of the living cell. The result is iatrogenic illnesses, the illnesses that arise through medical intervention. I would like to quote the extent of these iatrogenic illnesses: recently, American physicians in Boston determined that 36% of 815 patients had iatrogenic diseases, at the time they were examined when admitted to hospital; 9% of the cases were serious, and in 2% of the cases, the iatrogenic illness was probably a contributing factor to a fatal outcome. These are shocking figures. I believe that the best doctor is the one who is familiar with the fundamental principles of general biology and has an appreciation of the delicate and closely adapted, optimally matched, optimised organisation of cells. This physician utilises medicines very, very cautiously. These cases of poisoning by medicinal products can in part be traced back to the ill pressuring the doctor to prescribe medicine, since many patients incorrectly consider the doctor who most often prescribes to be the best doctor. Finally, I would like to mention that overmedication has been a problem for centuries - a problem that is very difficult to eliminate. Indeed even in Boston, where American doctors recently - in March 1981 - published these statistical data, right where we fight especially effectively - one would think - against overmedication, but success has been very limited so far. I would like to provide two quotes that show this to be an age-old problem. German physicist and philosopher Georg Christoph Lichtenberg, who lived from 1742 to 1799, once wrote, And a contemporary of his, Castello, wrote a pair of verses portraying a conversation between doctor and patient. The patient says, "Oh, dear doctor, how do you manage to remain so hearty and happy at such an advanced age?" And the doctor replies: "Simple, my dear child, I prescribe medicines for others, but I certainly never take them myself". And I hope doctors and physiologists acquire wisdom by studying the wonderfully optimised adaptation of living organisms to the environment and by studying general biology in the broadest sense. This is really the deeper reason, because we expect physicians to first thoroughly study the fundamental subjects, applied to the special case of Homo sapiens, before they get near a patient. I thank you.

Sir Hans Krebs (1981)

The Evolution of the Citric Acid Cycle and other Metabolic Pathways

Sir Hans Krebs (1981)

The Evolution of the Citric Acid Cycle and other Metabolic Pathways

Comment

Sir Hans Adolf Krebs was a German biochemist and a true specialist for elucidating cyclic metabolic pathways, i.e. cyclic series of chemical reactions in a cell. He is credited with the discovery of three such pathways, the ornithine cycle, the glyoxylate cycle and – most importantly – the citric acid or Krebs cycle. In the Krebs cycle, acetate, originating from the degradation of sugars or fatty acids, is further degraded to carbon dioxide, thereby yielding energy in the form of ATP and GTP molecules. The intermediates of the cycle are also important precursors for the synthesis of other biomolecules, such as amino acids. In total, the Krebs cycle accounts for two thirds of the oxidation of carbon compounds in most cells [1]. It thus appears to be fair to consider it the most important metabolic process altogether.In 1937, Krebs and his graduate student William A. Johnson attempted to publish their groundbreaking discovery in the journal Nature. Surprisingly, their article, which should turn out to be one of the most important biological papers of the 20th century, was rejected [2]. It eventually appeared in another journal, Enzymologia [3]. In the present lecture, which was given more than 40 years after this publication, Krebs takes a look back and considers a seemingly trivial question. Why is the citric acid cycle a cycle? Why is it not a linear pathway? Krebs claims to have been unable to answer this question, which was repeatedly asked by his students, until 1980, when he discussed the issue with his Oxford colleague, Jack Baldwin, a professor of organic chemistry and true specialist of reaction mechanisms.Baldwin suggested that the cyclic nature of the oxidation of acetate may have evolutionary reasons. Naturally, evolution would prefer the mechanism which yields most energy, that is most ATP molecules. Whereas a non-cyclic process, based on the direct oxidation of acetate, would be thinkable, it would have to involve several unfavorable reaction mechanisms, thus yielding less ATP. The intermediates of the citric acid cycle, on the other hand, allow for very efficient enzymatic reactions and thus ensure that the maximum possible amount of ATP molecules is generated from each acetate unit entering the cycle.In concluding his talk, Krebs expresses his concern about a tendency of overmedication in modern medicine. According to Krebs, physicians should always stay aware of the ultimate degree of optimization and efficiency Nature has achieved for metabolic processes. This quasi-perfect status quo makes it very unlikely, that a drug tampering with metabolism will ever change anything for the better, Krebs says. His statement, which is now more than 30 years old, could not be more topical: drug side-effects are estimated to be the fourth to sixth leading cause of death in the United States today [4]. The present lecture is the last of nine lectures Krebs gave in Lindau from 1960 to 1981. He passed away in Oxford only five months later, on November 22nd.David Siegel[1]B. Alberts et al., Molecular Biology of the Cell, Fifth Edition, 2008, Garland Science, New York, USA. [2]H. Kornberg, Nature Reviews Molecular Cell Biology 2000, volume 1, pages 225-228.[3]H. A. Krebs and W. A. Johnson, Enzymologia 1937, Volume 4, pages 148-156. [4]J. Lazarou et al., Journal of the American Medical Association 1998, volume 279,pages 1200-1205.

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