Under normal aerobic conditions, glycolysis proceeds through nine enzymatic reactions to produce pyruvate; under anaerobic conditions, pyruvate is converted by one additional enzymatic reaction to lactate. The latter has been considered a useless end product, of which tissues must be rid of, as many investigators, then, and even now, held it to be harmful. This description of the glycolytic pathway has stood unchallenged for more than six decades.
However, beginning in the s, studies in the fields of both muscle and brain energy metabolism have indicated that lactate is not a useless product of anaerobic glycolysis, but rather a potential important player in energy metabolism in these tissues and possibly others. The present chapter describes the key biochemical and physiological data both from the early days of research on carbohydrate metabolism and those gathered over the past three decades that have challenged the original, dogmatic layout of the glycolytic pathway.
Hopefully, this chapter will spur biochemists, physiologists and neuroscientists to consider the reconfiguration of glycolysis as proposed here and elsewhere.
This usually follows with the qualification that under aerobic conditions, the glycolytic pathway leads up to the tricarboxylic acid cycle TCA and the electron transfer chain ETC , the two biochemical processes responsible for capturing the majority of energy contained in glucose.
In contrast, under anaerobic conditions, such as those existing in working muscles, pyruvate is reduced to lactate. The elucidation of the glycolytic pathway was completed in , thanks mainly to studies by Meyerhof, Embeden, Parnas, Warburg, Neuberg and Gerty and Carl Cori.
It has been the first biochemical pathway to be elucidated, opening the door for future such puzzle solutions and to the field of biochemistry as we know it today. For those who are interested in refreshing their knowledge about the ten or so enzymatic steps of glycolysis and the coenzymes, substrates and products of these steps, any recent biochemistry textbook will do see also Figure 1A and B. Nevertheless, despite some uncertainties that have led to unproven assumptions about the role and function of the two alternative glycolytic end products, pyruvate and lactate, the glycolytic pathway has been accepted as originally proposed in The first nine reactions of glycolysis are summarily listed in Figure 1A.
These nine reactions end with pyruvate, the product suggested as the substrate for the mitochondrial TCA cycle under aerobic conditions. Since under anaerobic conditions mitochondrial respiration is halted, a 10th reaction was added to the original glycolytic pathway formulation where pyruvate is reduced to lactate by lactate dehydrogenase LDH, Figure 1B.
Hence, under anaerobic conditions, glycolysis was postulated to reach a dead-end point. A schematic illustration of the classic glycolytic pathway as originally perceived both under aerobic A and anaerobic B conditions. Under aerobic conditions, pyruvate is assigned as the end-product of the pathway, while under anaerobic conditions, lactate is the end product.
This is one of the main drawbacks of the classical aerobic glycolytic pathway. In , Brooks [ 3 ] published results showing that during prolonged exercise of skeletal muscle, lactate is both produced glycolytically and consumed oxidatively.
The finding that activated brain tissue produces lactate [ 5 ] should not have been that surprising, since it indicates that activated brain tissue resorts to non-oxidative energy production similar to activated muscle tissue.
However, the findings by both Brooks [ 3 ] and Schurr et al. Consequently, one must wonder why it took over four decades to produce results that challenge the dogma of two separate glycolytic pathways, aerobic and anaerobic. Alternatively, could it be that earlier findings in both muscle and brain tissues had already pointed at the possibility that lactate is more than just a useless end product of glycolysis, but for obscure reasons were ignored?
In a review article, Schurr [ 22 ] examined the history of carbohydrate energy metabolism from its earlier stages at the end of the nineteenth century to the elucidation of the glycolytic pathway in and beyond. That review has unearthed some intriguing findings, both about the scientists who were leading the field at the time and the interpretation of their own research data.
The scientific debate that ensued following the publications by Brooks [ 3 ] and Schurr et al. Sour milk, where lactic acid lactate was first discovered, sets the tone for what has become for years to come the negative trademark of this monocarboxylate. Once found in working muscle, lactate was immediately blamed for muscle fatigue and rigor. As early as , Fletcher [ 24 ] demonstrated that lactic acid he used 0. The higher the lactic acid concentration, the quicker the rigor mortis sets in.
Moreover, Fletcher and Hopkins [ 25 ] have shown that in the presence of oxygen, the survival of the excised muscle was prolonged and so did the acceleration of the disposal of lactate from it.
These researchers highlighted the recognition that the body has the means to rid itself from muscular lactate and that there is ample evidence that such disposal is most efficient under oxidative conditions.
Thus, the dogma of lactate as a muscular product responsible for fatigue and rigor, one that aerobic conditions enhance its disposal, was already well entrenched among scientists at the beginning of the twentieth century. It is still entrenched today among athletes and their coaches. Hill [ 7 , 8 ] went even further than Fletcher by suggesting that the role of oxygen in muscle contracture is twofold, to decrease the duration of heat production and to remove lactate from it.
Hill argued that the measured heat production of lactate oxidation was much lower than the calculated value of its complete combustion. It is somewhat perplexing that a scientist of the stature of Hill would argue that if lactate were a fuel, all the energy of its oxidation would be released as heat. The leading investigators in the field at the time actually concluded that lactate is a separate entity from the one that is oxidized during muscle respiration and which yields energy and CO 2.
Moreover, they held that the energy yielded in respiration is utilized for lactate disposal. By the s [ 28 , 29 ], the central theme of these studies and many others had been muscle tissue and its glycolytic formation of lactate. The process had been postulated to always be anaerobic and mainly through the breakdown of glycogen. In addition, when aerobic oxidation takes place, it occurs only after the muscle contracts and its main purpose is the removal of accumulated lactate and its accompanied acidosis.
The relationship between lactate and glycogen in muscle and, eventually, in other tissues, including brain, has been a complicating issue in the understanding of glycolysis. While the muscular conversion of glycogen to lactate is still in dispute today [ 30 ], both Nobel laureates had a long-lasting influence on this field of research.
Since the majority of scientists in the field of carbohydrate metabolism in those days studied muscle tissue, their interpretation of and opinions about the results of their studies greatly influenced those who studied carbohydrate metabolism of other tissues, especially brain. Thus, the small scientific community that investigated cerebral glycolysis in the late s and early s adopted the opinions of their peers in the field of muscle glycolysis and accepted the popular dogma, according to which, lactate is a useless end product that the brain eliminates via oxidation.
That concept stood against their own notion that the results of their studies could indicate lactate oxidative utilization by brain tissue. While Hill and Meyerhof were the leading scientists in the field of muscle carbohydrate metabolism in the s and s, E. Holmes was their counterpart in the field of cerebral carbohydrate metabolism. The latter was joined by his wife, B. First, they showed that brain carbohydrates are not the source of brain lactate; however, the brain is capable of forming lactate from added glucose [ 31 ].
In their second study, they determined that brain lactate levels fall when there was a fall in blood sugar level, which results in shortage of glucose in the brain [ 32 ]. In the third paper of the series, the Holmes found that brain tissue in room temperature or under anaerobic conditions does not exhibit a significant increase in lactate level or a significant fall in glycogen level, but that under aerobic conditions, lactate rapidly disappears, while glycogen level remains unchanged [ 33 ].
Thus, the Holmes established that glucose is the precursor of lactate in the brain and that under aerobic conditions, brain lactate content decreases. Additionally, these investigators showed that brain lactate is formed from glucose supplied by the blood and that its levels rise and fall with blood glucose levels, under both hypo- and hyperglycemic conditions.
Moreover, they showed that the diabetic brain is not different from the normal brain, where lactate formation and its removal under aerobic conditions are concerned [ 34 ]. By , Ashford joined Holmes and the two were able to demonstrate that the disappearance of lactate and the consumption of oxygen are correlated, which, in essence, indicates an aerobic utilization of lactate by brain tissue.
Furthermore, these investigators also showed that sodium fluoride NaF , the first known glycolytic inhibitor, blocked both glucose conversion to lactate and oxygen consumption. Holmes [ 35 ] showed in brain gray matter preparation that oxygen consumption was completely inhibited by NaF in the presence of glucose. However, when lactate was used instead of glucose, oxygen consumption was not inhibited by NaF. Consequently, Holmes concluded that the conversion of glucose to lactate must take place prior to its oxidation by brain gray matter.
These results and their straightforward conclusion have been completely ignored for over eight decades. This ignorance is especially glaring when one considers the fact that by the time the glycolytic pathway was elucidated in , Holmes and Ashford papers were already available for at least a decade [ 35 , 36 ] and should have been taken into account prior to the announcement of that elucidation. Hence, 76 years ago, we could have been presented with somewhat different view of the glycolytic pathway instead of the one in which, depending on the presence or absence of oxygen, ends up with either pyruvate or lactate, respectively.
Krebs and Johnson were careful to place a question mark following their suggestion that pyruvate is the TCA cycle substrate. Thus, the work by the Holmes couple [ 31 — 34 ], Ashford and Holmes [ 36 ] and Holmes and Ashford [ 41 ] on brain carbohydrate metabolism has been ignored and remained obscure even today, due mainly to habit of mind [ 23 ].
This habit of mind prevents many scientists from accepting more recent data that challenge the old dogma of a glycolytic pathway that has two possible outcomes, aerobic and anaerobic.
Nevertheless, we must not forget that in , both the fact that the TCA cycle enzymes are located in mitochondria and the role these organelles play in respiration were unknown. Also unknown at the time was the fact that mitochondria contain in their membrane the enzyme lactate dehydrogenase LDH , which can convert lactate to pyruvate [ 42 — 51 ]. Ignorance is understandable where the general public is concerned as both coaches and athletes continue, unabated, to blame lactic acid for muscle pain following anaerobic effort, even as recently as during the Rio Olympic games despite the fact that this claim has been refuted [ 52 ].
It is followed by the Krebs cycle and oxidative phosphorylation to produce ATP. In the first half of glycolysis, energy in the form of two ATP molecules is required to transform glucose into two three-carbon molecules. In the first half of glycolysis, two adenosine triphosphate ATP molecules are used in the phosphorylation of glucose, which is then split into two three-carbon molecules as described in the following steps.
The first half of glycolysis: investment : The first half of glycolysis uses two ATP molecules in the phosphorylation of glucose, which is then split into two three-carbon molecules. Step 1. The first step in glycolysis is catalyzed by hexokinase, an enzyme with broad specificity that catalyzes the phosphorylation of six-carbon sugars.
Hexokinase phosphorylates glucose using ATP as the source of the phosphate, producing glucosephosphate, a more reactive form of glucose. This reaction prevents the phosphorylated glucose molecule from continuing to interact with the GLUT proteins.
It can no longer leave the cell because the negatively-charged phosphate will not allow it to cross the hydrophobic interior of the plasma membrane. Step 2. In the second step of glycolysis, an isomerase converts glucosephosphate into one of its isomers, fructosephosphate. An enzyme that catalyzes the conversion of a molecule into one of its isomers is an isomerase.
This change from phosphoglucose to phosphofructose allows the eventual split of the sugar into two three-carbon molecules. Step 3. The third step is the phosphorylation of fructosephosphate, catalyzed by the enzyme phosphofructokinase. A second ATP molecule donates a high-energy phosphate to fructosephosphate, producing fructose-1,6-bisphosphate.
In this pathway, phosphofructokinase is a rate-limiting enzyme. This is a type of end-product inhibition, since ATP is the end product of glucose catabolism. Step 4. The newly-added high-energy phosphates further destabilize fructose-1,6-bisphosphate. The fourth step in glycolysis employs an enzyme, aldolase, to cleave 1,6-bisphosphate into two three-carbon isomers: dihydroxyacetone-phosphate and glyceraldehydephosphate. Step 5. In the fifth step, an isomerase transforms the dihydroxyacetone-phosphate into its isomer, glyceraldehydephosphate.
Thus, the pathway will continue with two molecules of a single isomer. At this point in the pathway, there is a net investment of energy from two ATP molecules in the breakdown of one glucose molecule.
So far, glycolysis has cost the cell two ATP molecules and produced two small, three-carbon sugar molecules. Both of these molecules will proceed through the second half of the pathway where sufficient energy will be extracted to pay back the two ATP molecules used as an initial investment while also producing a profit for the cell of two additional ATP molecules and two even higher-energy NADH molecules.
Step 6. The sugar is then phosphorylated by the addition of a second phosphate group, producing 1,3-bisphosphoglycerate.
Note that the second phosphate group does not require another ATP molecule. Here, again, there is a potential limiting factor for this pathway. If oxygen is available in the system, the NADH will be oxidized readily, though indirectly, and the high-energy electrons from the hydrogen released in this process will be used to produce ATP.
Step 7. In the seventh step, catalyzed by phosphoglycerate kinase an enzyme named for the reverse reaction , 1,3-bisphosphoglycerate donates a high-energy phosphate to ADP, forming one molecule of ATP.
This is an example of substrate-level phosphorylation. A carbonyl group on the 1,3-bisphosphoglycerate is oxidized to a carboxyl group, and 3-phosphoglycerate is formed. Step 8. In the eighth step, the remaining phosphate group in 3-phosphoglycerate moves from the third carbon to the second carbon, producing 2-phosphoglycerate an isomer of 3-phosphoglycerate.
The enzyme catalyzing this step is a mutase isomerase. Step 9. Enolase catalyzes the ninth step. This enzyme causes 2-phosphoglycerate to lose water from its structure; this is a dehydration reaction, resulting in the formation of a double bond that increases the potential energy in the remaining phosphate bond and produces phosphoenolpyruvate PEP.
Step Many enzymes in enzymatic pathways are named for the reverse reactions since the enzyme can catalyze both forward and reverse reactions these may have been described initially by the reverse reaction that takes place in vitro, under non-physiological conditions.
Glycolysis starts with one molecule of glucose and ends with two pyruvate pyruvic acid molecules, a total of four ATP molecules, and two molecules of NADH.
Two ATP molecules were used in the first half of the pathway to prepare the six-carbon ring for cleavage, so the cell has a net gain of two ATP molecules and 2 NADH molecules for its use.
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