Krebs Cycle Takes Place in What Part of the Cell

K

In Dictionary of Energy (Second Edition), 2015

Krebs cycle the cycle of chemical reactions that are the major source of energy in living organisms. [Described by Hans Adolf Krebs.]

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Krebs cycle The Krebs cycle, also known as the citric acid cycle or the tricarboxylic acid cycle, is one of the most important reaction sequences in biochemistry. Not only is this series of reactions responsible for most of the energy needs in complex organisms, the molecules that are produced in these reactions can be used as building blocks for a large number of important processes, including the synthesis of fatty acids, steroids, cholesterol, amino acids for building proteins, and the purines and pyrimidines used in the synthesis of DNA. Fuel for the Krebs cycle comes from lipids (fats) and carbohydrates, which both produce the molecule acetyl coenzyme-A (acetyl-CoA). This acetyl-CoA reacts in the first step of the eight step sequence of reactions that comprise the Krebs cycle, all of which occur inside mitochondria of eukaryotic cells. While the Krebs cycle does produce carbon dioxide, this cycle does not produce significant chemical energy in the form of adenosine triphosphate (ATP) directly, and this reaction sequence does not require any oxygen. Instead, this cycle produces NADH and FADH₂, which feed into the respiratory cycle, also located inside of the mitochondria. It is the respiratory cycle that is responsible for production of large quantities of ATP and consumption of oxygen. In addition, the respiratory cycle converts NADH and FADH₂ into reactants that the Krebs cycle requires to function. Thus, if oxygen is not present, the respiratory cycle cannot function, which shuts down the Krebs cycle. For this reason, the Krebs cycle is considered an aerobic pathway for energy production.

Clarke Earley

Kent State University

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Biochemical Reactions and Enzyme Kinetics

John D. Enderle PhD , in Introduction to Biomedical Engineering (Third Edition), 2012

8.5.2 Krebs Cycle

The Krebs cycle involves a series of enzyme catalyzed reactions that reduce the acetyl portion of acetyl coenzyme A in the mitochondrial matrix, as shown in Figure 8.25. The Krebs cycle continuously recycles, reusing the substrates and enzymes with an overall reaction given by

Figure 8.25. Overview of the Krebs cycle.

(8.103) $\begin{array}{l}Acetyl-CoA+3NA{D}^{+}+FAD+2H_{2}O+ADP+P⟶\\ CoA-SH+2C{O}_2+FAD{H}_2+ATP+3\left(NADH+H^{+}\right)\end{array}$

The reaction begins with the joining of acetyl-coenzyme A with oxaloacetate and water to form citrate and is given by

(8.104) $\begin{array}{l}AC+H_{2}O+CS+O\stackrel{B_1}{⟶}ACOCS\stackrel{K_1}{⟶}CT+CoA-SH+CS\\ {\dot{q}}_{AC}=-B_1{q}_{AC}{q}_{H_{2}O}{q}_{CS}{q}_O+J_{AC}\\ {\dot{q}}_{ACOCS}=B_1{q}_{AC}{q}_{H_{2}O}{q}_{CS}{q}_O-K_1{q}_{ACOCS}\end{array}$

where $J_{AC}$ is the flow of acetyl coenzyme A based on Eq. (8.102).

The next step involves the reaction of citrate with the enzyme aconitase to create isocitrate, which is given by

(8.105)

The third step involves the reaction of isocitrate and $NA{D}^{+}$ with the enzyme isocitrate dehydrogenase to create $\alpha -\text{ketoglutarate},\left(NADH+H^{+}\right)\text{and}C{O}_2$ , which is given by

(8.106)

The fourth step involves the reaction of $\alpha -ketoglutarate,NA{D}^{+}\text{and}CoA$ with the enzyme $\alpha -\text{ketoglutarate dehydrogenase}$ to create $\text{succinyl}-\text{CoA}$ , $\left(NADH+H^{+}\right)$ , and $C{O}_2.$ This reaction is given by

(8.107) $\begin{array}{l}KG+KGH\stackrel{B_4}{⟶}KGKGH\\ KGKGH+NA{D}^{+}+CoA-SH\stackrel{K_4}{⟶}SC+KGH+\left(NADH+H^{+}\right)+C{O}_2\\ {\dot{q}}_{KG}=K_3{q}_{ITID}{q}_{NA{D}^{+}}-B_4{q}_{KG}{q}_{KGH}-K_{-3}{q}_{KG}{q}_{ID}{q}_{NADH+H^{+}}{q}_{C{O}_2}\\ {\dot{q}}_{KGKGH}=B_4{q}_{KG}{q}_{KGH}-K_4{q}_{KGKGH}{q}_{NA{D}^{+}}{q}_{CoA}\end{array}$

The fifth step involves the reaction of $\text{succinyl}-\text{CoA},\text{A}\text{D}\text{P},\text{and}P$ with the enzyme $\text{succinyl}-\text{CoA}$ $\text{synthetase}$ , to create $\text{succinate},CoA\text{and}\text{A}\text{T}\text{P}.$ This reaction is given by

(8.108)

The sixth step involves the reaction of succinate and $FAD$ with the enzyme succinate dehydrogenase to create fumarate and FADH ₂. This reaction is given by

(8.109)

The seventh step involves the reaction of fumarate and $H_{2}O$ with the enzyme fumarase to create malate. This reaction is given by

(8.110)

The last step involves the reaction of malate and $NA{D}^{+}$ with the enzyme malate dehydrogenase to create oxaloacetate and (NADH + H ⁺). This reaction is given by

(8.111)

The final equation combines the oxaloacetate from the first reaction with that in the last reaction:

(8.112) ${\dot{q}}_O=K_8{q}_{MTMTD}{q}_{NA{D}^{+}}-K_{-8}{q}_O{q}_{MTD}{q}_{NADH+H^{+}}-B_1{q}_O$

It should be clear that this reaction continually recycles and that it requires two cycles to process each glucose molecule.

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BIOCHEMISTRY OF MUSCLE MITOCHONDRIA*

E.J. DE HAAN , ... E.M. WIT–PEETERS , in Physiology and Biochemistry (Second Edition), 1973

E The Respiratory Chain

In the oxidation reactions of the Krebs cycle, the hydrogen atoms (or the electrons derived from them) do not react directly with oxygen, but pass through a series of hydrogen or electron carriers, the respiratory chain. The present picture of the respiratory chain is shown in Fig. 2.

Fig. 2. The respiratory chain in mitochondria.

The primary hydrogen acceptor for the oxidation of pyruvate and α-ketoglutarate is the lipoic acid covalently bound to one of the proteins of the corresponding keto–acid dehydrogenase complex. Hydrogens from the reduced lipoic acid are transferred to NAD⁺ in a reaction catalyzed by lipoamide dehydrogenase. NAD⁺ is also the hydrogen acceptor for the oxidation of a number of substrates, including malate, isocitrate, and l-3-hydroxyacyl-CoA, each oxidoreduction being catalyzed by a specific dehydrogenase.

There are two NADP⁺-linked dehydrogenases in heart muscle mitochondria catalyzing the oxidative decarboxylation of isocitrate and malate; threo-D_s-isocitrate: NADP⁺ oxidoreductase (decarboxylating) (EC 1.1.1.42) is present both in the mitochondrion (80–85%) and in the extramitochondrial compartment (Goebell and Pette, 1967). Brdiczka and Pette (1971) have shown that in heart muscle, "malic" enzyme (malate: NADP⁺ oxidoreductase (decarboxylating) (EC 1.1.1.40)) occurs predominantly in the mitochondrial fraction (68–86% of the total cellular activity, depending on the species). The transfer of hydrogens from NADPH to NAD⁺ is catalyzed by a transhydrogenase (see Section IV,B).

Another flavoprotein in the respiratory chain transfers hydrogens from NADH to Q:NADH dehydrogenase. Q is also the hydrogen acceptor for the flavoprotein-catalyzed oxidation of succinate and of acyl-CoA. The oxidation of QH₂ by molecular oxygen involves the transfer of electrons rather than hydrogens (or hydride ions), as occurs in the first part of the respiratory chain. Electrons are transferred successively to cytochrome b, cytochrome c₁, and cytochrome c. The enzyme catalyzing the oxidation of reduced cytochrome c by oxygen is cytochrome oxidase; it contains two heme groups, a and a₃, and two copper atoms (Beinert et al., 1970).

Considerable attention has been paid to the occurrence of spectrally distinct species of cytochrome b in the respiratory chain (for reviews, see Slater, 1971; Van Dam and Meijer, 1971). These different species can be detected under different states of energization of the mitochondria.

Several inhibitors of the respiratory chain and of the oxidative phosphorylation coupled with it (Section III,F) are known. Some practical information concerning their application in biochemical research is summarized by Slater (1967).

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The Use of Synthetic Biology Tools in Biorefineries to Increase the Building Blocks Diversification

André T.V. Hermann , ... Fernando Segato , in Advances in Sugarcane Biorefinery, 2018

3.8.3 Succinic Acid

This dicarboxylic acid is part of the Krebs cycle, and can be obtained from microbiological fermentation and used as building blocks to synthesize several polymers of commercial importance, such as polybutylene succinate (PBS) that shows characteristics similar to polyethylene (PET). In combination with diamines, putrescine, and cadaverine, this molecule can be used to produce materials based on 100% biological nylon. Moreover, it can also be used as a chemical platform to be transformed by simple chemical processes into 1,4-butanediol (1,4-BDO), gamma-butyrolactone (GBL), tetrahydrofuran (THF), and N-methylpyrrolidone (NMP) (Choi et al., 2015; Tsuge et al., 2016).

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Recent Progress in the Solid-State NMR Studies of Biomineralization

Tim W.T. Tsai , Jerry C.C. Chan , in Annual Reports on NMR Spectroscopy, 2011

4.2 OCP incorporated with succinate

Earlier biochemical studies had reported that the Krebs cycle in mitochondria are closely associated with the intramitochondrial precipitation of electron-dense mineral granules, ¹³⁸ which have been suggested as ACP containing OCP-carboxylate components. ¹³⁹ Previous studies have shown that succinate ions (OOCC₂H₄COO²⁻) can be incorporated into the lattice of OCP, ^139–142 leading to the formation of the OCP-succinate (OCPS) compound. ¹³⁹ Consequently, OCPS prepared in vitro could serve as a model system to mimic the biomineralization process in mitochondria. The XRD patterns of OCPS and OCP are very similar except that the characteristic peak of OCP at 2θ equal to 4.751 is shifted to 4.181 in OCPS, which is interpreted as the elongation of the d-spacing of the (100) plane from 1.86 to 2.13   nm. As described previously for OCP, ^102,105 the PO₄ ³⁻ groups at sites P2 and P4 may have a strong tendency to react with the neighbouring water molecule to form HPO₄ ²⁻. That is, the number of the hydrogen phosphate groups is more than what implied in the chemical formula of OCP because of the change in the spectroscopic parameters of the phosphorus species at P4 and/or P2. Not surprisingly, the same phenomenon is observed for OCPS, which implies that the loss of crystal water during the formation of OCPS may have caused a change in the hydrogen bonding environment of other phosphorus sites. ¹⁴³ In particular, the similarity of their τ _CP values suggests that the "excessive" P1 may come from P2 and/or P4. In any case, it is doubtless that the site occupation at P5 is significantly reduced upon the incorporation of succinate. From the literature, ⁹⁹ it is known that each unit cell of OCP contains two formula units of Ca₈(HPO₄)₂(PO₄)₄⋅   5H₂O. Consequently, the structure of OCPS can be qualitatively described as a compound comprising one succinate molecule per unit cell of OCPS, in which one of the two P5s will be replaced by succinate ions. ¹⁴³ Such replacement is accompanied by a significant loss of structural water. The hydrogen bonding of the remaining P5 with water molecules would be weakened considerably as indicated by the significant increase of its chemical shift span. Such incorporation of the succinate ions must be driven by the enthalpy gain of certain well-defined interactions, leading to a uniform change in the periodicity of the cell dimension.

The ³¹P MAS spectrum of OCPS has been assigned by ³¹P homonuclear DQ spectroscopy. On the basis of the deconvolution data of the ³¹P MAS spectrum and the thermogravimetric analysis results, the molecular formula of OCPS was determined to be Ca_7.81(HPO₄)_1.82(PO₄)_3.61(succinate)_0.56⋅ zH₂O, where z  ≤   0.5. ¹⁴³ When succinate ions are incorporated to form the OCPS lattice, mainly the phosphorus species at the P5 site will be displaced. The stability of OCPS is significantly higher than OCP with respect to the hydrolysis reaction at high pH condition. Apparently, the succinate ions will considerably dampen the dynamics of the water molecules within the hydration layer, rendering the relocation of the phosphate ions within the hydration layer very difficult. Previously, it has been shown that the transformation of OCP to HAp upon an increase in pH is accomplished by structural rearrangement in the hydration layer, followed by the concatenation of the hence formed HAp sublattices. ¹⁰⁴ Together with the results of OCPS, it is clearly shown that the structure of the hydration layer of OCP is rather versatile and is playing the key role in the structural transformation of OCP. We note in passing that some organic molecules such as citrate ions can also be incorporated into synthetic calcite by coprecipitation. ¹⁴⁴

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Mitochondria

In Division of Labor in Cells (Second Edition), 1970

THE METABOLISM OF CARBOHYDRATES

The role of mitochondria in oxidative phosphorylation has already been mentioned and their role in the metabolism of carbohydrates through the presence in their substance of enzymes concerned with the Krebs cycle and the cytochrome system has been indicated. We should now consider the problem of carbohydrate metabolism and the role that mitochondria and other parts of the cytoplasm play in it.

Carbohydrate metabolism is extremely important for cell synthesis and is the main source of energy for cell activities. Before attempting to localize the various activities of carbohydrate metabolism in the actual parts of the cell, we should consider briefly what the metabolism of carbohydrates involves. There are two types of metabolism, anaerobic and aerobic. The anaerobic route is demonstrated very well by muscle, and most of the information on this type of metabolism of carbohydrates has been obtained by studies of this tissue.

The result of anaerobic metabolism is the production of lactic acid and the liberation of a good deal of CO₂. However, although we think in terms of anaerobic metabolism for muscle, we have to realize that muscle itself has a first-class blood supply that appears to be increased by various physiological mechanisms when muscle is forced to do work and that muscle in the process of contraction uses a rather surprisingly large amount of oxygen.

Lactic acid has been shown to accumulate in muscle extracts and in isolated muscles kept under anaerobic conditions. If we consider the accumulation of lactic acid in an animal in vivo, we find that after moderate work the accumulation of lactic acid goes up slightly but remains at a pretty steady level. However, if strenuous work is done, then the amount of lactic acid goes up extremely steeply and slowly comes back to normal after the work has ceased. The reason for this is that under normal circumstances muscle can obtain oxygen fast enough to reoxidize the lactic acid as rapidly as it is formed and only a small amount of lactic acid accumulates. However, it is possible for muscle to do more work than it can supply oxygen for and it can do this by oxidizing carbohydrates anaerobically and so accumulating lactic acid. Eventually this lactic acid has to be oxidized with the aid of atmospheric oxygen but it can be stored and reoxidized later and this can take place over a longer period of time. Some of the lactic acid is converted into glycogen in the liver and the rest is oxidized. The fact that it is possible to accumulate lactic acid and slowly oxidize this after the work is finished provides a mechanism by means of which an "oxygen debt" can be produced. Under aerobic circumstances it is not lactic acid that is formed in the metabolism of carbohydrates but pyruvate. However, this does not accumulate and it is oxidized almost as rapidly as it is formed—as we shall see in a minute there is a very complicated system for oxidizing this pyruvate. The only time when pyruvic acid does accumlate in the tissues is in the absence of vitamin B₁ or thiamine, a fact that was demonstrated years ago in Oxford University by R. A. Peters. Under a process known as oxidative decarboxylation with the aid of the cocarboxylase (thiamine pyrophosphate), it yields acetate, carbon dioxide, and lactate. It has been stressed that this reaction is fundamentally of an oxidative nature and is called oxidative decarboxylation, an important process both in carbohydrate and protein metabolism.

We have mentioned the production of pyruvate but have not yet considered the process by means of which this compound is produced—this process is known as glycolysis and it represents the preliminary stage in the metabolism of carbohydrates. Glycolysis is, in effect, the reverse of photosynthesis. In photosynthesis the energy of sunlight is used to combine CO₂ and water into carbohydrates; in the process of glycolysis, the glucose that is formed from carbohydrates such as glycogen and other polysaccharides is converted into pyruvate and then into CO₂ and water with the liberation of energy. We can express this as C₆H₁₂O₆ + 6O₂ = H₂O + 6CO₂ + energy. This oxidation of glucose to give CO₂, water, and energy is not a single step but involves a very large number of steps of considerable complexity. During the various steps, small packets of energy are released at a rate at which the cell can use them, whereas if there were a sudden explosive release of energy by the oxidation of glucose, the cell would probably not be able to use this relatively large amount of energy in a coordinated way and a good deal of it would probably be wasted.

The first stages of glycolysis involve the phosphorylation of glucose and this is done with the aid of ATP as follows (the enzyme concerned in this process is placed above the arrow):

$\text{glucose}+\text{ATP}\stackrel{\text{hexokinase}}{\to }\text{glucose}-6-\text{phosphate}+\text{ADP}$

Hexokinase is not just one enzyme, there are in fact a number of hexokinases that catalyze phosphorylation of hexoses. The phosphorylation of glucose results in the transferring of a high-energy phosphate from the ATP to glucose and so to form a phosphate ester that is poor in energy—this type of reaction is called an exergonic reaction and is essentially irreversible. It is of interest that one of the properties of glucose-6-phosphate that differs from glucose is the fact that the phosphate ester has difficulty in penetrating cell membranes whereas glucose itself, apparently, crosses without any difficulty, and it has been suggested that this hexokinase reaction is one way in which glucose can be locked in a cell. It is also an essential prerequisite for the resynthesis of glycogen.

Many things can happen to glucose apart from phosphorylation and ultimate conversion into CO₂ and water via the glycolytic and Krebs cycle system. Among these are its dehydrogenation by a glucose dehydrogenase to form gluconic acid. This is done by a diphosphopyridine nucleotide (DPN)-linked (codehydrogenase) reaction.

An International Commission has recently altered the names of some of these coenzymes—for example, DPN now becomes nicotinanide adenine dinucleotide (NAD); when it has an additional third phosphate group, it is known as NADP, which was called triphosphopyridine nucleotide (TPN) before the Commission changed its name. This compound, though closely related to NAD, cannot replace it in the reactions in which it forms an integral part. Likewise, NAD cannot replace NADP in the reactions in which it is a constituent. NADP seems to play an electron-transport role in a smaller number of dehydrogenases than NAD. Glucose-6-phosphate dehydrogenase is a typical enzyme for which NADP acts as an electron acceptor. The reduced forms of these coenzymes are indicated by NADH and NADPH.

In mammals, it is believed that this is not a usual pathway for glucose to follow. If glucose can be locked into position in a cell by being converted into a phosphate, it is obvious that there must be in existence a mechanism that can release it again since it needs, for instance, to be fed from the liver periodically into the bloodstream to keep the blood glucose level at a relatively constant figure. This is carried out by a specific enzyme, glucose-6-phosphatase,

$\text{glucose}-6-\text{phosphate}+{\text{H}}_2\text{O}\stackrel{\text{G}-6-\text{Pase}}{\to }\text{glucose}+{\text{PO}}_4.$

This glucose-6-phosphatase is probably present in all tissues which release glucose from cells, but it does not appear to occur in skeletal muscle.

Glucose-6-phosphate may be converted into glucose-1-phosphate, in other words the phosphate is simply shifted around from the 6-position to the 1-position on the glucose molecule. This change of position of the phosphate group is catalyzed by an enzyme known as phosphoglucomutase, and the reaction changing the phosphate group from one part of the molecule to the other is an easily reversible reaction. The formation of glucose-1-phosphate from glucose-6-phosphate is a stage in the synthesis of glycogen, for instance, if glucose is accumulating in a cell it is converted to glucose-6-phosphate and then to glucose-1-phosphate. The molecule of glucose-1-phosphate then polymerizes into glycogen. The reverse process can occur—glucose-6-phosphate can be obtained from glycogen by first producing glucose-1-phosphate and then converting it into glucose-6-phosphate. We have, however, diverted a little from the direct line of our story of the glycolytic cycle. After the formation of glucose-6-phosphate from glucose, the next stage in the glycolytic cycle is the conversion of glucose-6-phosphate into fructose-6-phosphate. This reaction is readily reversible and is catalized by an enzyme called phosphohexose isomerase.

Fructose-6-phosphate may also be formed directly from fructose, and it is known that an enzyme, fructokinase, which is present in brain and muscle can produce this effect. The next step is the further phosphorylation of fructose-6-phosphate; another phosphate group is added in the 1-position to give fructose 1-6-diphosphate. The enzyme responsible for this is phosphofructokinase and the reaction is carried out with the aid of ATP:

$\text{frucotose}-6-\text{phosphate}+\text{ATP}\stackrel{\text{PFkinase}}{\to }\text{frucotose1}-6-\text{diP}+\text{ADP}$

This reaction is exergonic (heat producing).

Fructose-1-6-diphosphate is also known as hexose diphosphate and in the next stage this is broken down by what is described as the "aldolase" reaction into three phosphorylated compounds—triosephosphates. The first of these is ketose triosephosphate, the second is dihydroxyacetone phosphate, the third is phosphoglyceraldehyde. The triosephosphate can be converted into phosphoglyceric acid; dihydroxyacetone phosphate can be converted into phosphoglyceraldehyde or alternatively it can be reduced to α-glycerophosphate with the aid of DPNH (NADH) and α-glycerophosphate dehydrogenase. This reaction is potentially important for the synthesis of lipids since from the α-glycerophosphate, phosphatidic acid can be synthesized, and phosphatidic acid can be the starting point for the synthesis of lecithin, cephalin, and also of fats. Phosphoglyceric acid, with the aid of the enzyme enolase, becomes converted into phosphoenol-pyruvic acid.

Pyruvic acid penetrates cell membranes very well and can thus leave the cell and in theory can be distributed to any cell in the body.

Conversely, all the steps we have mentioned can be reversed and pyruvate can be converted back into glucose-6-phosphate. When pyruvic acid is reduced it gives lactic acid. Lactic acid itself can be converted back to pyruvic acid. Liver cells are capable of reversing the whole glycolytic series of reactions and can produce glucose and glycogen again from lactic acid. Muscle can reverse lactic acid to glucose-6-phosphate and glucose-1-phosphate and glycogen but cannot produce nonphosphorylated glucose.

In these first stages of carbohydrate metabolism, a certain amount of energy is liberated but this is not much more than about one-tenth of the total amount of energy which is produced by the production of CO₂ and water from glucose. The glycolytic part of the cycle is the least energy-producing part.

In addition to its conversion into lactic acid or its oxidation, pyruvic acid can be converted to alanine, an amino acid, by the process of transamination. Thus here one can see a link between carbohydrate and protein metabolism.

If there is ample oxygen, the pyruvic acid produced by this first stage of carbohydrate metabolism can be oxidized. This is a complex process in which in the first stage the pyruvic acid is converted by the process of oxidative decarboxylation into acetyl coenzyme A and CO₂. Thiamine pyrophosphate (cocarboxylase) is an essential enzyme for this process.

Acetyl coenzyme A is a two-carbon substance and it condenses with oxaloacetic acid which is a four-carbon dicarboxylic acid to yield a six-carbon tricarboxylic acid, namely, citric acid (or citrate). The enzyme catalyzing this is called the "condensing enzyme." Thus starts a series of changes that ultimately lead to the formation of CO₂ and water. The production of citric acid is followed by the loss and recapture of water, and it becomes converted to cis-aconitic acid or cis-aconitate (with the aid of aconitase, glutathione, and ferrous iron), which on further hydration is converted into isocitric acid. This production of isocitric acid can, however, take place directly without the production of free cis-aconitate (cis-aconitic acid). This compound then loses hydrogen and thereby becomes oxidized to oxalosuccinic acid or oxalosuccinate (this reaction is catalyzed by isocitric dehydrogenase) and decarboxylation turns it into α-ketoglutaric acid (the enzyme responsible is oxalosuccinic decarboxylase plus oxidized manganese). α-Ketoglutaric acid is decarboxylated and then oxidized by the loss of two hydrogen atoms to succinic acid (the enzyme concerned is α-ketoglutaric dehydrogenase). More recent studies have shown that this process is more complicated—the decarboxylation and oxidation of α-ketoglutarate is carried out by four coenzymes: thiamine pyrophosphate, lipoic acid, coenzyme A, and nicotinamide adenine dinucleotide (NAD). An intermediary substance succinyl coenzyme A is first produced from α-ketoglutarate and succinate is then liberated from the succinyl coenzyme A. Succinic acid is also oxidized by the loss of H₂ (with the help of succinic dehydrogenase) to fumaric acid. The actual oxidation of succinate occurs by the transfer of two electrons to ferricytochrome B, which is a member of the electron-transport chain. Succinic dehydrogenase is the enzyme that catalyzes this transfer. This enzyme contains iron and a flavin coenzyme. The latter by the addition of the elements of water becomes converted by fumarase into malic acid, and the malic acid by dehydrogenation catalyzed by the enzyme malic dehydrogenase is converted in oxaloacetic acid, and there we are back at the beginning of the cycle. The oxaloacetic acid (oxaloacetate) is ready to combine with another molecule of acetyl coenzyme A to produce citric acid once more. During this whole process three molecules of CO₂ and five molecules of H₂ are given off.

It can be seen that quite a complex series of reactions take place in what has been called the "tricarboxylic" or "citric-acid cycle," or the "Krebs cycle." All three terms are applicable.

The final combination of the hydrogen atoms liberated by the Krebs cycle with oxygen is brought about by the cytochrome system. It is of interest that cytochrome is a protein that contains a form of heme, an iron containing pigment that is also present in hemoglobin. It is probably more widely distributed than any other type of heme protein since it occurs in the cells of all organisms that use oxygen, irrespective of whether they are animals or plants or whether in the case of animals they are vertebrates or invertebrates, or protozoa.

In the cytochrome system a variety of compounds and enzymes are involved. Cytochrome oxidase is a widely distributed enzyme since its occurrence is comparable in distribution to cytochrome. It has not yet been obtained pure, and it has been found to be bound to the insoluble material when cells are homogenized and spun down. This is because it is associated with the mitochondria. There is also another compound involved called flavoadenine dinucleotide (FAD for short) which is associated with various proteins to form a variety of different enzymes. A flavin containing enzyme was first isolated and called a "yellow ferment" as long ago as 1932 by Warburg and Christian. Flavoproteins are usually associated with various metals; molybdenum, copper, and iron are three that have been found to be necessary for their function. It is not quite clear where the metal atoms are situated on the enzyme molecule. There are two types of riboflavin-containing enzymes, one type is an electron acceptor from reduced DPN (NAD) or TPN (NADP), and one type can transfer electrons either to oxygen or to the cytochromes, whereas the other type of flavoprotein accepts electrons directly from metabolites. Among the important flavoproteins is cytochrome reductase which comes in two types—one for reduced DPN (DPNH) (NAD, NADH) and the other for reduced TPN (TPNH) (NADP, NADPH). The oxidation of these two compounds DPNH and TPNH (NADH and NADPH) in the cell can thus be carried out by the cytochrome system.

Now we are in a position to describe the next stages that take place in the oxidation of glucose. The five pairs of hydrogen atoms that are passed down to the cytochrome system from the Krebs cycle are combined with the cytochrome and result in its reduction. During this process the hydrogen atoms dissociate to protons and electrons, and the electrons are handed along an electron-transport chain and eventually reduce atmospheric oxygen. The enzyme concerned in the reduction of cytochrome is cytochrome reductase, the flavoprotein already mentioned. Cytochrome is then oxidized with the aid of cytochrome oxidase, which removes the hydrogen atoms. They become combined with atmospheric oxygen to produce water and thus the long journey of oxidation of carbohydrates is done, five molecules of water and three molecules of CO₂ being produced from each molecule of pyruvate. There are, in fact, a series of cytochromes arranged in a chain, and the electrons are passed along these prior to being combined with atmospheric oxygen. Although the course of oxidation may seem tedious and involved to the reader of the preceding pages, all these reactions take place in a flash. Reference back to the section on mitochondria will remind the reader that the electron-transfer chain also goes through coenzyme Q, or "ubiquinone" a compound closely related to vitamin K.

An analysis of oxygen uptakes of various tissues in the body gives an indication of the degree to which their cells are metabolizing. As a matter of interest, it might be noted that of the tissues examined, retina and kidney had the highest metabolism and liver was next, then the rate decreased progressively from adrenal, lung, bone marrow, diaphragm, heart, lymph nodes, skeletal muscle, skin to eye lens, which had the lowest level of oxygen consumptions. One important point to remember is that the complex of reactions described above is localized to a great extent in the mitochondria.

One of the important functions of this oxidative cycle just described is its relationship to phosphorylation. We have described earlier the work of Green who showed that oxidative phosphorylation could take place without the whole Krebs cycle occurring, but in intact mitochondria the whole cycle normally goes through and phosphorylation is an important by-product of these reactions. This process of phosphorylation results in the production of high-energy phosphate esters, such as ATP.

Since ATP is one of the principal energy-containing compounds of the body, it is very important in the whole energy cycle of the cell. It is, for example, the main source of energy in musclar contraction and for many of the synthetic processes of other cells.

Although the relation between ATP formation and the respiratory chain has been widely accepted scientifically, Dr. H. Rottenberg of Brooklyn College, at the meeting of the American Society for Experimental Biology held in Atlantic City (1969), has suggested that this relationship is more complicated than it may seem. He described his views as a "chemiosmotic theory" and a number of workers have held similar views. In this theory, instead of cell respiration being directly coupled to phosphorylation (ATP production), the respiratory process generates a proton flux across the inner membranes of the mitochondria and this "proton pump" is geared directly into ATP synthesis.

This briefly is the story of carbohydrate metabolism in the cell. What we want to try to do now is to demonstrate where this complex of activity is situated in the living cell. It appears that most of the processes of respiration and glycolysis actually take place in the cytoplasm and mitochondria. Some years ago (1941), the present author and R. J. Allen demonstrated that zymohexase, which is really a complex of two enzymes and is concerned with the splitting of hexose diphosphate (the aldolase reaction), was localized in the cytoplasm of the cell and in the case of muscle fibers in between the fibrils in the sarcoplasmic material rather than in any of the formed elements. It is noted too that the results of differential centrifugation of cell homogenates have demonstrated that most of the enzymes responsible for the glycolytic cycle have been found to be present either in the supernatant or in "microsomes" and those concerned with the cytochrome system and Krebs cycle are localized specifically in the mitochondria. Now, since the microsomes mostly represent fragments of the endoplasmic reticulum, we can assume that most of the enzymes found in them can also be found in the membranes of the endoplasmic reticulum. In addition to the aldolase complex (see Fig. 57), which has been demonstrated to be present in the supernatant, it has been found that phosphorylase and phosphoglucomutase and glucose-6-phosphate dehydrogenase have also been found in the supernatant and glycolysis has in fact been found to take place in this fluid—a fact that confirms that all the glycolytic enzymes necessary for this process must be present there. However, glucose-6-phosphatase has been found not to be localized at all in the supernatant but exclusively in the microsomes (fragmented endoplasmic reticulum). There is some evidence too that hexokinase is present in the microsomes. On the other hand, DPNH (NADH) and DPN (NAD) and TPN-(NADP-) linked cytochrome c reductases that have been found in high concentration in mitochondria have also been found to be present in the microsomes. It might be possible to suggest from these facts a tentative scheme that would help us to understand to some extent the relationship between the reactions occurring in the cytoplasm itself and those in the mitochondria.

FIG. 57. Aldolase preparation of intestine showing diffuse reaction in the smooth muscle.

Figure and legend from Allen and Bourne, J. Exptl. Biol. 20, 61, 1943.) Copyright © 1943

Let us start off by assuming that some glucose has passed across the cell membrane. Perhaps it was taken in by pinocytosis as has been suggested by a number of authors or, possibly, if and when the membranes of the endoplasmic reticulum are continuous with the outside of the cell, it may simply have passed up the cisternae between the membranes of the reticulum and entered into the cell through the membranes of this reticulum. Let us assume that the glucose has passed into the cell by the process of pinocytosis. Then, with hexokinase present in the cytoplasm, it can be converted into glucose-6-phosphate and subsequently run through the cycle to pyruvate. At this point the enzyme system associated with the mitochondria comes into play. If the glucose has to pass across the cell membrane or across the membranes of the endoplasmic reticulum, it should also be able to do this without too much difficulty. If any glucose-6-phosphate should occur in the cisternae, it will probably be able to pass freely into the cytoplasm because of the presence of glucose-6-phosphatase in the membrane. It may be that there is hexokinase in the fluid within the cisternae and that, perhaps, all the glucose there (if any) is first phosphorylated and then relèased in a timed fashion into the cytoplasm through the action of glucose-6-phosphatase in the membranes (see Fig. 58). Perhaps this is a way of controlling the feeding of the carbohydrate fuel into the furnace. It will be remembered that glucose-6-phosphate passes cell membranes with difficulty unless there is the appropriate hydrolytic enzyme on the membrane. The glucose, once into the cytoplasm by whatever route, can be synthesized into glyogen or it can be brought to pyruvate by the enzymes of the glycolytic cycle that are known to occur in the cytoplasm. Once pyruvate is prepared, it is changed into acetyl coenzyme A and is ready for the next stage. For this it has to come into contact with the mitochondrial membrane. Now, it has been mentioned that a great proportion of the enzymes concerned with the Krebs tricarboxylic acid cycle are localized in the mitochondrial membranes or particles associated with them and since acetyl coenzyme A appears to be the most commonly used fuel of this system, it is at this point that the mitochondria largely take over the final oxidative stages. Thus, as far as we can tell at the moment either in the cytoplasm of the cell itself or in the endoplasmic reticulum, glycolysis occurs with the production of either acetyl coenzyme A or pyruvic acid and then these compounds are converted by the mitochondrial enzymes into CO₂ and water, thus completing the oxidation of carbohydrates. We do not know at what stage these compounds are fed to the mitochondria. They must go largely to the mitochondria because these organelles contain the greatest proportion of the Krebs cycle enzymes—it is these latter that complete the oxidative breakdown of carbohydrate that was set into motion by hexokinase. If under normal conditions we have a large number of mitochondria occupying the cell, we can assume, other things being equal, that oxidation should be proceeding at a fast rate because of the large mitochondrial surface area available. However, where one gets a large increase of number of mitochondria as, for example, in starvation, this may be compensatory hypertrophy and not indicative of increase in oxidative activity. We do not know whether the nature of the mitochondrial surface varies from time to time but we know that the surface area varies. Mitochondria, for instance, fragment from the filamentous and rodlike condition to the granular state under a variety of circumstances. These include mechanical damage to the cells, rough handling, influence of bacteria and other toxins, as a result of anesthesia and anoxia, and so on. It can be shown mathematically that there is a greater surface area available the more fragmented the mitochondria become. Because of this either the pyruvate and/or acetyl coenzyme A can feed more rapidly into or become attached in larger amounts to the mitochondria when they are in a fragmented condition simply because there is a greater surface area. Thus the mitochondria by virtue of their ability to fragment and re-form into the filamentous condition can act as a throttle controlling the rate of aerobic metabolism in the cell. They may also have an additional means of doing this, a sort of fine control. Since it is fairly certain that enzyme molecules are aligned along the cristae, these structures represent another surface where reactions can take place, and reduction in the size and number of cristae would also have a throttling effect on the metabolism or synthesis due to mitochondria. This can be seen in operation in the case of a mitochondrion that has accumulated a good deal of fat or other product of chemical reactions. In such a case, the cristae are reduced or absent altogether as if metabolic processes are brought to a virtual standstill because of the accumulation of reaction products. This brings us to another process of control, a chemical method known as "feedback," which will be discussed shortly.

FIG. 58. Schematic representation of relationship of endoplasmic reticulum to carbohydrate metabolism.

From Siekewitz, "Regulation of Cell Metabolism," Ciba Foundation Symp., Churchill, London, 1959.) Copyright © 1959

Siekewitz believes that both glycogen synthesis and breakdown take place at the surface between the cytoplasm and the ergastoplasmic membranes in association with the enzymes present at those surfaces. He points out, however, that only glucose-6-phosphatase and DPNH (NADH) and TPNH (NADPH) cytochrome c reductases have been found to be associated with the microsome fraction, but he believes that the site of effective action of the other enzymes might be at the interface between the membranes and the matrix of the cytoplasm. He suggests that hexokinase might be activated at the membrane surface of the endoplasmic reticulum. Other cofactors such as glucose-1-6-diphosphate and adenosine monophosphate possibly also bind their appropriate enzymes to the E.R. membranes. (DPNH and TPNH are believed to act as coenzymes for glycolysis in the early stages of glucose oxidation.)

Siekewitz has also discussed certain biochemical aspects of the control of glucose metabolism.

First of all he points out that, if the concentration of glucose in the lumen of the endoplasmic reticulum is in equilibrium with the glucose concentration in the blood (this assumes at least temporary continuity between the lumen or cisternae of the E.R. and the exterior of the cell), there exists then a mechanism whereby the glucose level in the blood would definitely affect intracellular glucose equilibrium. Thus a reduced production of glucose from the diet would lead to reduction of glucose in the blood and this would cause a reduction of the amount of glucose in the fluid within the endoplasmic reticulum. The latter result would lead to increased phosphatase activity which would cause an increase in the breakdown of glycogen (e.g., in the liver cells) and a production of glucose, which would pass out from the endoplasmic reticulum into the bloodstream.

Siekewitz points out that hexokinase, phosphoglucomutase, and phosphorylase might have their activity enhanced if they were attached to the endoplasmic reticulum membranes. In the case of phosphorylase, active phosphorylase B has to undergo a conversion to active phosphorylase A before it can have any catalytic effect. An enzyme phosphorylase B-kinase carries out this conversion by phosphorylating the enzyme in the presence of ATP. Siekewitz suggests that this kinase may be part of the membrane of the endoplasmic reticulum and that the activating process for phosphorylase in the cell might consist of moving it out of the cytoplasm onto the site of the E.R. membranes. Siekewitz points out that it is possible that hormones control this type of movement of enzymes within the cell and its internal membranes. At this point one might remember the work quoted earlier by Brandes and the present author in which it was demonstrated that shifts of acid phosphatase activity took place from the Golgi apparatus to the nucleus in ventral-lobe prostate cells following castration and that acid phosphatase activity was restored to the Golgi apparatus following implantation of the male sex hormone. Also of interest are the further studies of Brandes in which he has demonstrated that in castrated animals there is a rearrangement of the membranes of the endoplasmic reticulum. These studies demonstrate a definite morphologicobiochemical effect on the part of the male sex hormone. Siekewitz suggests that the hormones concerned with carbohydrate metabolism do not act directly on the enzyme as such but bring it and the substrate and various cofactors together at a suitable surface and then complex them together there (see Fig. 58). Figures 59A, B, C, D, E, and F show the possible origin of an enzyme in the nucleus and its passage to the endoplasmic reticulum.

FIG. 59. Glucose-6-phosphatase in rat spinal ganglion neurons. (A) Note reaction restricted to nucleolus; (B) note reaction in nucleus, nucleolus still positive appears outside the nucleus and in contact with the cell membrane; (C) strong reaction in nucleolus, rest of nucleus and cytoplasm give moderate diffuse reaction; (D) moderate reaction in nucleus, stronger in cytoplasm, intense perinuclear reaction; (E) nucleolus and nucleus slight to negative reaction, reaction in cytoplasm appears to be associated with the endoplasmic reticulum; (F) completely negative nucleus and nucleolus. Cytoplasm filled with large negative vacuoles; cytoplasm between the vacuoles is strongly positive. These results suggest a cyclic metabolic activity in the spinal ganglion cells. It is possible that glucose-6-phosphatase is synthesized in the nucleolus and passed first to the nucleus and then to the cytoplasm where it becomes associated with the endoplasmic reticulum. The presence of large synthetic products in the cytoplasm suggest the participation of the glucose-6-phosphatase in some synthetic process. This product is not glycogen, but some of our protein and fat preparations show moderate sized droplets which could be identical with the vacuoles.

Preparations and photographs by H. B. Tewari, Dept. of Anatomy, Emory Univ.)

Other enzymes may be localized on the endoplasmic reticulum, e.g., 5 nucleotidase.

The nucleus, as will be described later, appears to be enclosed in a fold of the double membrane of the endoplasmic reticulum and not to have a membrane of its own. Since the E.R. spaces might be connected directly to the exterior of the cell, it is possible that glucose coming from outside the cell could be converted into glucose-6-phosphate in the E.R. lumen and thus be prevented from passing through the E.R. membrane. Thus it could pass along all the ramifications of the E.R. canals and come in direct contact with the nuclear fold of the reticulum. If this membrane contains glucose-6-phosphatase (and a number of our histochemical studies suggest that it does, see Fig. 59), glucose could penetrate through into the nucleus without having to pass through the substance of cytoplasm at all. Histochemical preparations show that the glucose-6-phosphatase reaction in a particular histological section is not always positive for all nuclei in the section, and it is of interest that Siekewitz has pointed out that enzymes may be present or activated at the E.R. membrane only when the glucose concentration reaches a critical level.

It is of interest that histochemical study of the cells of many organs demonstrates the fact that many dephosphorylating enzymes as well as glucose-6-phosphatase show an association with nuclear membranes—perhaps here lies the mechanism whereby even low levels of glucose-6-phosphate could penetrate easily through into the interior of the nucleus. The endoplasmic reticulum could provide a pathway straight to the nucleus that would prevent glucose from coming into contact with or passing through the cytoplasm where it could be attacked by glycolytic enzymes. This may be the mechanism whereby a supply of glucose to the nucleus is ensured. Glucose could also be supplied to the nucleus from the cytoplasm by the process of glycolysis. In this case, glucose being formed from glycogen as glucose-6-phosphate would be dephosphorylated and pass through the E.R. membrane and into the lumen where possibly it would be rephosphorylated to prevent its passing back again and would thus move along the lumen to the nucleus. Hence the nucleus could get its glucose directly from the cytoplasm via the endoplasmic reticulum or directly from the outside (the latter, however, only if a direct connection really exists.)

In many cells the mitochondria can also be seen to be completely surrounded by endoplasmic reticulum. It is possible that the mitochondria themselves obtain their glycolytic fuel directly as a result of glucose passing through the E.R. membranes undergoing glycolysis there and the glycolytic products feeding directly to the mitochondria. Some authors have suggested that mitochondria are themselves no more than diverticula of the endoplasmic reticulum.

The control of the rate of different types of metabolism, particularly respiration, in the cell can depend on two main factors. First, a structural factor that brings into apposition the appropriate reactants and varies with the extent of the surfaces available for these processes to take place, and, second, it may also depend on some chemical feedback where an excessive production of one kind of compound inhibits its continued production or slows down further synthesis. Alternatively, the metabolism in any one particular direction may be affected by the absence of a limiting amount of some specific substance or compound in the reaction chain. In the case of glucose, there is a complex system of control of its production and use. For example glucose-6-phosphate activates glycogen synthetase, which is one of the enzymes concerned in the synthesis of glycogen, the effect of this being to stimulate the storage of excess glucose as glycogen. At the same time if glucose-6-phosphate accumulates in some tissues, it inhibits the enzyme hexokinase that would normally phosphorylate glucose, thus the net effect of the accumulation of glucose-6-phosphate is to cut down on the phosphorylation of more glucose. This process is known as "negative feedback."

Sir Hans Krebs ^* in a recent article entitled "Rate Limiting Factors in Cell Respiration," discussed the control of energy utilization and pointed out that, in unicellular organisms, energy can be obtained directly from oxidation if air is present but if air is not present then anaerobic fermentation takes its place and energy is obtained by this source. In this instance it is the supply of air or oxygen that regulates which of these mechanisms comes into use, and oxygen is, in fact, the "rate-limiting factor." In higher animals the ability to undergo fermentation or anaerobic oxidation is still present and can be particularly well demonstrated in muscle. Krebs pointed out that the chemical systems in the cell that are concerned with the function of regulation are all fairly simple reactions but that there is an elaborate interlocking of these reactions. By this he means that the individual reactants may take part not only in more than one reaction but in very many different processes. One of the difficulties in sorting out such a complex of activity is that not all the component reactions of this elaborate interlocking series are known and we are dealing with a heterogeneous system in which there are many varied membranes and different spatial arrangements of the various reactants. He also points out that regulation is probably a matter of reaction velocities, some of which will be accelerated and some slowed down and the question that has to be decided is the degree to which any of these are rate limiting. Krebs illustrates this point by considering the amount of oxygen used by 4-ml sheep heart homogenate, which contained about 10% of tissue (see tabulation). Thus one can demonstrate that the addition

Substrate added	O2 (μmole) used by 4 ml suspension
None	17
Pyruvate	26.1
Succinate	32.6
1-Lactate	21.6
Citrate	20.8
α-Oxyglutarate	25.4
Fumarate	19.1
Acetate	21.1
Glycogen	15.4

of glycogen adds nothing to the oxygen uptake so the amount of glycogen present is not a limiting factor in this system. The same applies if glucose is added instead of glycogen. On the other hand, when acetate, pyruvate, or other intermediates of the tricarboxylic cycle are added, there is an appreciable increase in the rate of oxygen uptake. The fact that the oxygen uptake in this system can be increased if suitable substrates are added to the mixture demonstrates that the electron-transport system from DPNH (NADH) to oxygen is not being used to its full capacity; thus it cannot be the factor that is limiting the uptake of oxygen. Certain special substrates that are known to reduce either DPN (NAD) or flavoprotein seem to be able to increase oxygen consumption. Thus the limiting factor appears to be that the mechanism for the transport of hydrogen from DPN (NAD) or flavoprotein is not being used to its full capacity if these special substrates are not present. The limiting step, therefore, is really the first stage in the electron transport system. If it is found that a particular substrate increases the rate of respiration, this is due to the fact that the substrate reacts more readily or easily with DPN (NAD) or flavoprotein than any endogenous substrate already present. This is the reason why pyruvate or α-ketoglutarate or succinate are responsible for the stimulation of respiratory rate in the experiment quoted. However, even if we accept this we are still faced with the identification of the factor that decides the rate of reaction between substrate and DPN (NAD) or flavoprotein.

Studies with dinitrophenol, which uncouples oxidative phosphorylation from respiration, are of interest and help to throw light on this problem.

Perhaps we should first say a word or two about this action of dinitrophenol. The uncoupling of oxidative phosphorylation from respiration has been compared to putting a car into neutral gear and still leaving the engine running. If the respiratory activities are regarded as the engine and the phosphorylation as the process of making the car go, then dinitrophenol uncouples the engine from the transmission of the car, the engine continues to turn but the car does not move; in the cell the respiration goes on quite happily but no ATP is formed. Normally ATP is formed from ADP and inorganic phosphate, and thus the factor that limits the rate of oxygen consumption and oxidation of pyruvate in the normal system such as we have described is not really the amount of enzymes present but actually the level of either ADP or inorganic phosphate. In the experiments carried out by Krebs, inorganic phosphate was present in a fairly good concentration and further quantities added to the system did not stimulate respiration. Therefore it is almost certain that the limiting factor must be the amount of ADP which is available. These experiments demonstrate the type of chemical control that a single compound can exert on a whole chain of reactions. One should also remember that another mechanism, a structural one that controls the rate of respiration, is the rate at which glucose can enter the cell and this can be hormonally controlled, although the method of action of the hormone is not exactly known. It is of interest that one of the factors that probably affects the rate at which glucose can enter the cell (if, in fact, the endoplasmic reticulum is continuous with the outside of the cell) is the degree of complexity of the endoplasmic reticulum. If this structure develops many ramifications, as presumably it seems able to do in certain cells such as spermatocytes, as demonstrated by Fawcett, then the surface area available for glucose to enter into the cytoplasm of the cell and so be metabolized is enormously increased or, conversely, it may be decreased by a reduction in complexity of the reticulum.

To return to the subject of respiration and ATP formation, the reaction for this process can be given as follows:

${\text{C}}_6{\text{H}}_{12}{\text{O}}_6(\text{g}\text{l}\text{u}cos\text{e})+6{\text{O}}_2+38\text{A}\text{D}\text{P}+38{\text{H}}_3\text{P}{\text{O}}_4\to 6\text{C}{\text{O}}_2+44{\text{H}}_2\text{O}+38\text{A}\text{T}\text{P}$

This is the general reaction and is a summary of all the complex intermediatary reactions that in the end simply produce carbon dioxide, water, and ATP. Since, as Slater and Houlsman have pointed out, cells contain only relatively small amounts of ADP, as soon as it is all converted into ATP the process of respiration will stop—ADP is thus the limiting factor. However, when the cell is stimulated to do work there is a breakdown of ATP according to the formula given by Slater and Houlsman,

$38\text{ATP}+38{\text{H}}_2\text{O}\to 38\text{ADP}+38{\text{H}}_3{\text{PO}}_4\to \text{work}$

and, since ADP is now being re-formed, respiration can go on so long as there is some left to be resynthesized into ATP. The addition of further ADP will, of course, keep respiration going.

It is of interest that, if mitochondria that have been separated from the cell by differential centrifugation are permitted to stand for some hours at room temperature, the phenomenon of uncoupling (which can also be brought about by dinitrophenol) of the oxidative phosphorylation system from respiration takes place. This type of mitochondrial preparation is described as "aging" mitochondria, and it is tempting to speculate whether in senescing tissues there may not be a progressive uncoupling of respiration from oxidative phosphorylation or a progressive hydrolysis of ADP so that less and less of this becomes available for synthesis of ATP. It has been possible to isolate from mitochondria that have been aged in this way a heme compound that will actually produce this uncoupling reaction. It has been described and given the name "mitochrome" and is fundamentally a pigment. However, there appears to be a lipid component in this "mitochrome" heme-protein preparation that is the actual factor responsible for uncoupling, and the heme protein of the mitochrome is not, in fact, the uncoupling factor at all. Mitochrome is very similar in structure and form to cytochrome and is probably derived from it. Certain unsaturated fatty acids such as oleic acid are also found to be active as uncoupling agents, and the lipid isolated from the mitochrome particle also appears to contain an unsaturated fatty acid. The fact that an uncoupling agent can be produced in vitro this way is of considerable importance since it seems possible that the formation of such a substance in mitochondria might take place in vivo and may itself function as a controlling agent for respiration and oxidation. See Fig. 60, a diagram showing the relation of cell structures to function.

FIG. 60. Differential distribution of biochemical activities in the liver cell according to Novikoff. A, oxidized substrate; aa, amino acid; AH, reduced form of substrate; ADP, adenosine diphosphate; AM P, adenosine monophosphate; AR, agranular reticulum (smooth E.R.); ATP, adenosine triphosphate; ATPase, adenosinetriphosphatase; BC, bile canaliculus; CMP, cytosine monophosphate; D-DPN, desamido diphosphopyridine nucleotide; DPN, diphosphopyridine nucleotide; DNA, deoxyribonucleic acid; D-NMT, deamido nicotinic acid mononucleotide; DB, peribilary dense bodies; Er, ergastoplasm; GA, Golgi apparatus; GTP, guanosine triphosphate; L, lysosomes; Mit, mitochondria; Nu, nucleus; P, inorganic phosphate; P-ase, phosphatase; P-RNA, particle ribonucleic acid; PP, inorganic pyrophosphatase; PS, protein synthesis; PV, pinocytosis vacuoles; RNA, ribonucleic acid; S-RNA, soluble ribonucleic acid.

From Novikoff, Am. J. Med. 29, 102, 1960.) Copyright © 1960

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Fluorinated Compounds in Enzyme-Catalyzed Reactions

V. Prakash Reddy , in Organofluorine Compounds in Biology and Medicine, 2015

5.4 Block Effect on Enzyme Inhibition: Aconitase Inhibition in the Citric Acid Cycle

The citric acid cycle (also called as Krebs cycle or tricarboxylic acid cycle) takes place in the mitochondria and is an integral part for the generation of adenosine triphosphate (ATP). In the citric acid cycle, 36  mol of ATP are formed from a single glucose molecule, and reduced nicotinamide adenosine diphosphate and other organics are also formed, which serve as intermediates for the biosynthesis of amino acids (e.g., glutamate is synthesized from α-ketoglutaric acid, an intermediate in the citric acid cycle).

Fluoroacetate is the starting compound for the biosynthesis of the fluorinated compounds in certain soil bacteria. However, fluoroacetyl-CoA (18), derived from the fluoroacetate, also competes with acetyl-CoA in its citrate-synthase-catalyzed reaction with oxaloacetate to form (2R, 3R)-2-fluorocitrate (20), which undergoes aconitase-catalyzed dehydration to give the 2(R)-2-fluoro-cis-aconitic acid (21) (in analogy to the normal citric acid cycle, in which cis-aconitic acid is formed from acetyl-CoA). Whereas the hydration of cis-aconitic acid gives isocitric acid, the next intermediate in the normal citric acid cycle, 21 undergoes S_N2′ type of hydroxylation–defluorination to give (R)-hydroxy-trans-aconitic acid (22), which no longer can participate in the citric acid cycle and, moreover, it acts as an irreversible inhibitor of the aconitase enzyme, resulting in shutting down of the citric acid cycle, and thereby exerting cytotoxicity in humans and other mammals. ⁴¹ In order to establish the mechanism of the aconitase inhibition by the secondary metabolite 22, a single-crystal X-ray structure determination was undertaken. The single-crystal structure of this enzyme–inhibitor complex revealed the tight binding of 22 to the aconitase at the active site. ⁴¹ His101, His147, and His167 have strong hydrogen bonding interactions with the substrate carboxylate moieties, and the resulting protonated His101 also forms hydrogen bond to the inhibitor hydroxyl group, which, in turn, forms a hydrogen bond to Asp165 (Figure 21). Thus the enzyme inhibitory action of the toxic metabolite 22 is not through its covalent binding to the amino acid residues at the enzyme active site, but rather through its tight fitting in, and thereby blocking, the active site. ⁴¹

Figure 21. Single-crystal X-ray structure of 4-hydroxy-(E)-aconitate (22; shown as stick model with oxygens in red) bound to the active site of aconitase at 2.05   Å resolution (generated using UCSF Chimera software; pdb:1FGH); The metabolite 22 binds to the active site very tightly but not through covalent binding, and is stabilized by extensive hydrogen bonding interactions with the His101, His147, and His167 residues; thus, it inactivates the aconitase and shuts down the citric acid cycle. ⁴¹

Importantly, the 2(S)-2-fluoro-cis-aconitic acid, (enantiomer of 21) would not be able to undergo the similar S_N2′ type of hydroxylation–defluorination and therefore would not be able to inhibit the aconitase enzyme. The citrate-synthase-catalyzed reaction of oxaloacetate with Z-enolate-CoA (Z-19), followed by aconitase catalyzed dehydration would give the 2(S)-2-fluoro-cis-aconitic acid, whereas E-enolate-CoA (E-19) gives predominantly 21. The citrate synthase converts the fluoroacetyl-CoA (18), stereoselectively (98:2 diastereoselectivity) to E-19, resulting in the cytotoxicity associated with the fluoroacetate (Figure 22). Using ab initio quantum mechanics/molecular mechanics modeling Mulholland and coworkers ⁴² have rationalized the high stereoselectivity of the citrate synthase (i.e., the stereoselective formation of the E-19): the computed enzyme–substrate complex for the E-19 is about 2   kcal/mol more stable than that for Z-19, which is also in accord with the estimated Δ(Δ^{G ǂ}) value based on the experimentally observed ∼98:2 diastereomeric ratio of the 2(R)- and 2(S)-2-fluorocitrates.

Figure 22. (A) Conversion of acetyl-CoA to citrate in the citric acid cycle and (B) conversion of fluoroacetyl-CoA (18) to (R)-4-hydroxy-(E)-aconitate (22), which inhibits aconitase and thereby shuts down the citric acid cycle.

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Carbon metabolism

Kim Gail Clarke , in Bioprocess Engineering, 2013

5.2.2 Tricarboxylic acid cycle

The TCA ⁵ cycle provides a common pathway for the ultimate catabolism of carbohydrates and fats, which, through its oxidative aerobic nature, generates ATP mainly ⁶ via oxidative phosphorylation for anabolic processes. The fuel for the TCA is acetyl-CoA, a molecule which plays a pivotal role in sugar and fat metabolism. In fat catabolism, acetyl-CoA is formed during the β-oxidation of fats (Section 5.2.3) when fatty acids are fragmented into their acetyl-CoA components. In carbohydrate metabolism, acetyl-CoA is linked to glycolysis via the conversion of pyruvate to the thioester acetyl-CoA by incorporation of CoA (Figure 5.5). The conversion is catalysed by the pyruvate dehydrogenase complex in a series of steps during which pyruvate is decarboxylated with the release of CO₂ and subsequently dehydrogenated with the reduction of NAD⁺

Figure 5.5. Tricarboxylic acid cycle

The TCA cycle is initiated with the condensation of the acetyl-CoA with oxaloacetate to form citrate. The citrate is converted to isocitrate via addition of water across the double bond of cis-aconitate, the isocitrate is dehydrogenated to oxalosuccinate and the oxalosuccinate is decarboxylated to α-ketoglutarate. The subsequent conversion of α-ketoglutarate to succinate is mediated via the thioester, succinyl-CoA. Succinyl-CoA is first formed by decarboxylation and reduction of α-ketoglutarate, with the amalgamation of CoA, catalysed by the α-ketoglutarate dehydrogenase complex. The conversion of the succinyl-CoA to succinate features the single substrate level phosphorylation in the TCA cycle when the high energy bond in the succinyl-CoA is transferred to GDP, and subsequently to ADP, to form ATP with the incorporation of inorganic phosphate and release of CoA. Succinate is then reduced to fumarate (here FAD, rather than NAD⁺ is the primary molecule as the energy released is not sufficient to include the NAD⁺ step). Hydration of fumarate across the double bond yields malate, which on subsequent dehydrogenation leads back to oxaloacetate and the close of the TCA cycle.

The series of consecutive reactions during the TCA cycle forms reduced species (NADH and FADH₂), producing three NADH and one FADH₂ per acetyl-CoA molecule. NADH and FADH₂ then enter the respiratory chain where oxygen is incorporated as the final electron acceptor and inorganic phosphate taken up by ADP to produce ATP and H₂O as in Equations 5.6 and 5.7.

[5.6] $3\mathrm{ADP}+3\mathrm{Pi}+1/2{\mathrm{O}}_2\to 3\mathrm{ATP}+4{\mathrm{H}}_2\mathrm{O}\left(\mathrm{per}\mathrm{NADH}\right)$

[5.7] $2\mathrm{ADP}+2\mathrm{Pi}+1/2{\mathrm{O}}_2\to 2\mathrm{ATP}+3{\mathrm{H}}_2\mathrm{O}\left(\mathrm{per}{\mathrm{FADH}}_2\right)$

So, the energy balance for three NADH and one FADH₂ yields Equation 5.8.

[5.8] $11\mathrm{ADP}+11\mathrm{Pi}+2{\mathrm{O}}_2\to 11\mathrm{ATP}+15{\mathrm{H}}_2\mathrm{O}$

Now including the substrate level phosphorylation, the energy balance becomes Equation 5.9.

[5.9] $12\mathrm{ADP}+12\mathrm{Pi}+2{\mathrm{O}}_2\to 12\mathrm{ATP}+15{\mathrm{H}}_2\mathrm{O}$

During the oxidation of acetyl-CoA there is a net usage of two H₂O per molecule acetyl-CoA and a net production of two CO₂ and one Co A. So the overall equation for the complete oxidation of one molecule of acetyl-CoA can be written as Equation 5.10.

[5.10] $1\mathrm{acetyl}-\mathrm{CoA}+12\mathrm{ADP}+12\mathrm{Pi}+2{\mathrm{O}}_2\to 1\mathrm{CoA}+12\mathrm{ATP}+13{\mathrm{H}}_2\mathrm{O}+2{\mathrm{CO}}_2$

However, one NADH was also produced and one CO₂ liberated in the decarboxylation of pyruvate to acetyl-CoA, so incorporating the energy derived from oxidation of NADH (Equation 5.6) and the liberation of CO₂, the overall equation for the complete oxidation of one molecule of pyruvate can be written as Equation 5.11.

[5.11] $1\mathrm{pyruvate}+15\mathrm{ADP}+15\mathrm{Pi}+21/2{\mathrm{O}}_2\to 15\mathrm{ATP}+17{\mathrm{H}}_2\mathrm{O}+3{\mathrm{CO}}_2$

The overall energy balance from glucose under aerobic conditions, however, also has to incorporate the energy produced during glycolysis. Under aerobic conditions, the NADH produced during glycolysis will enter the oxidative phosphorylation chain, each NADH producing three ATP according to Equation 5.6. Two NADH are produced per glucose during glycolysis to yield an additional six ATP per glucose molecule. This, in addition to the net two ATP per molecule of glucose produced by substrate level phosphorylation during glycolysis, gives an overall balance of glucose to pyruvate under aerobic conditions (noting that two H₂O are produced per glucose during glycolysis) according to Equation 5.12.

[5.12] $1\mathrm{glucose}+8\mathrm{ADP}+8\mathrm{Pi}+{\mathrm{O}}_2\to 2\mathrm{pyruvate}+8\mathrm{ATP}+10{\mathrm{H}}_2\mathrm{O}$

Combining the contributions from glycolysis (Equation 5.12) and the TCA cycle (Equation 5.11) under aerobic conditions yields 38 ATP for the complete oxidation of one molecule of glucose to CO₂ and H₂O (Equation 5.13).

[5.13] $1\mathrm{glucose}+38\mathrm{ADP}+38\mathrm{Pi}+6{\mathrm{O}}_2\to 2\mathrm{Pyruvate}+38\mathrm{ATP}+44{\mathrm{H}}_2\mathrm{O}+6{\mathrm{CO}}_2$

The 38 ATP produced from glucose under aerobic conditions (Equation 5.13) is in stark contrast to the two ATP produced from glucose (Equations 5.4 and 5.5) by anaerobic fermentation. Clearly, glucose utilisation is far more energy efficient when reduced compounds are not formed.

An understanding of the underlying principles of the metabolism and energy generation is always important from a process viewpoint, especially when dealing with facultative microorganisms ⁷ in aerobic bioprocesses. Poor mixing can lead to pockets of depleted oxygen in large scale bioreactors. During passage through the anaerobic pockets, microbial metabolism will change from aerobic to anaerobic with consequent alteration of yields and productivities.

Arguably, the most well documented industrial example of the impact of underlying metabolism on process performance is in the production of the facultative yeast, S. cerevisiae, the mainstay of the baking and brewing industries. Efficient glucose utilisation provides optimum performance for growth of S. cerevisiae so process conditions should prevent glucose being diverted to ethanol and consequent reduction of ATP per molecule of glucose. However, under high concentrations of glucose, the TCA cycle enzymes are inhibited and energy production takes place by substrate level phosphorylation only, despite adequate oxygen supply. ⁸

To maximise the carbon conversion to yeast, a process strategy known as fed-batch (see Section 7.4) is used. Here the glucose is fed to the process at a rate that maintains the glucose concentration below the inhibitory level. This manipulation of process strategy to optimise process kinetics is a classic example of the synergism between life science and engineering where the success of the process depends on both an understanding of fed-batch process design equations and the regulation of enzyme synthesis which underpins the microbial behaviour.

The major function of the TCA cycle is to provide energy in the form of ATP in anabolic pathways. In addition, the TCA cycle also serves as a pool of intermediates and/or precursors for anabolic functions, e.g. amino acid biosynthesis. ⁹ However, if TCA cycle intermediates are used in this way, they have to be replenished by other reactions for energy generation to continue. These reactions are called anaplerotic reactions. One of the most important anaplerotic reactions is the reversible carboxylation of pyruvate to oxaloacetate at the expense of ATP which facilitates the start of gluconeogenesis (Section 5.3.2).

In some microorganisms, the TCA cycle is modified to eliminate those reactions which release CO₂ . This modified pathway, called the glyoxylate shunt, functions to conserve carbon. Conservation of carbon is particularly important in microorganisms which grow only on fats where acetyl-CoA serves not only to provide energy, but also as a carbon source. In the glyoxylate shunt, the isocitrate splits into glyoxylate and succinate. The glyoxylate then combines with acetyl- CoA to form malate which re-enters the TCA cycle. The shunt through glyoxylate eliminates two steps in the TCA cycle which liberate CO₂, hence carbon is conserved.

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Bacterial Metabolism–Coupled Energetics

R.S. Prakasham , B. Sudheer Kumar , in Microbial Electrochemical Technology, 2019

2.1.2.2.3 Reverse Tricarboxylic Acid Cycle

The reverse tricarboxylic acid cycle (rTCA) is also known as reverse Krebs cycle or the reverse citric acid cycle. In this cycle a sequence of metabolic pathways operate to produce carbon compounds (energy rich compounds) from carbon dioxide and water, and hence it is considered to be an alternative to photosynthesis or fixation of inorganic carbon in the reductive pentose phosphate cycle or reductive carboxylation [35]. In general, in TCA cycle, organism generates energy via the oxidation of acetate generated from either carbohydrate or protein or lipid using terminal electron accepter. In rTCA, the citric acid cycle pathway runs in opposite direction to synthesize carbon compounds of interest utilizing numerous ATP molecules. The enzymes, unique to reverse TCA, include pyruvate:ferredoxin (Fd) oxidoreductase (acetyl-CoA   +   CO₂  +   2Fd_red  +   2H⁺  ⇌   pyruvate   +   CoA   +   2Fd_ox), ATP citrate lyase (ACL, acetyl-CoA   +   oxaloacetate   +   ADP   +   P _i   ⇌   citrate   +   CoA   +   ATP), α-ketoglutarate:ferredoxin oxidoreductase (succinyl-CoA   +   CO₂  +   2Fd_red  +   2H⁺  ⇌   α-ketoglutarate   +   CoA   +   2Fd_ox), and fumarate reductase (succinate   +   acceptor   ⇌   fumarate   +   reduced acceptor). Among them, ATP citrate lyase is the key regulatory enzyme of this cycle. There are numerous anaerobic organisms that utilize a cyclic reverse TCA cycle, and the best example includes organisms Chlorobium thiosulfatophilum (classified under Thermoproteus) which are characterized as a hydrogen–sulfur autotroph [35]. Analysis of carbon flux of anoxygenic green sulfur bacterium, Chlorobaculum tepidum, revealed that rTCA cycle in this bacterium is active. Tang et al. [36] reported that the rTCA cycle is active during autotrophic and mixotrophic growth, whereas the flux from pyruvate to acetyl-CoA is very low; acetyl-CoA is synthesized through the rTCA cycle and acetate assimilation, whereas pyruvate is largely assimilated through the rTCA cycle; and acetate can be assimilated via both the RTCA and the oxidative TCA cycle. rTCA cycle mainly requires electron donors and often times, bacteria use inorganic compounds such as hydrogen, sulfide, or thiosulfate or minerals for this purpose [35,37]. It is considered that this rTCA is the main metabolic pathway during prebiotic early-earth conditions and hence is of great interest in the research of the origin of life. The differences between TCA and rTCA cycle is exemplified in Table 2.1.5.

Table 2.1.5. Metabolic Reactions of TCA and rTCA

Step	TCA Cycle	rTCA Cycle
1	Oxaloacetic acid   +   acetyl   ∼   CoA   –>   citric acid   +   HS   ∼   CoA	Oxaloacetic acid   +   NADH   +   H +–>   malate   +   NAD +
2	Citric acid—>   cis-aconitate   +   H2O	Malate   –>   fumarate   +   H 2 O
3	Cis-aconitate   +   H2O   –>   isocitrate	Fumarate   +   FADH 2–>   succinate   +   FAD
4	Isocitrate   +   NAD+  –>   oxalosuccinate   +   NADH   +   H+	Succinate   +   ATP   –>   succinyl   ∼   coA   +   ADP   +   Pi
5	Oxalosuccinate –>   α-ketoglutarate   +   CO2	Succinyl   ∼   coA   +   NADH   +   H+  –>   α-ketoglutarate   +   Acetyl   ∼   CoA   +   NAD+
6	α-Ketoglutarate   +   acetyl   ∼   CoA   +   NAD+  –>   succinyl   ∼   coA   +   NADH   +   H+	α-Ketoglutarate   +   NADH   +   H+  –>   isocitrate   +   NAD+  +   CO2
7	Succinyl   ∼   coA   +   GDP   +   Pi   –>   succinate   +   GTP	Isocitrate   –>   citrate
8	Succinate   +   FAD   –>   fumarate   +   FADH 2	Citrate   –>   oxaloacetic acid   +   acetyl   ∼   CoA +   ATP   +   Pi
9	Fumarate   +   H 2 O   –>   malate
10	Malate   +   NAD +   –>   oxaloacetic acid   +   NADH   +   H +

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Yeast-Based Biofuel Cells

YV Hubenova , in Encyclopedia of Interfacial Chemistry, 2018

Subcellular origin of electrons

It is recognized that the electrons transferred to the anode originate from glycolysis, Krebs cycle, or pentose phosphate pathway, PPP. The electrons and protons generated during the substrate oxidation are transferred on intracellular electron acceptors like NAD ⁺. The reduced molecules (NADH) donate electrons in respiration chains (ETC) while creating a proton gradient used for ATP production meeting cellular energetic and biosynthetic needs (growth and division). Despite the presence of different redox molecules in the cell, the main cellular electronophore is NADH. The NADH/NAD⁺ pair has the most negative redox potential (−   0.320   V vs. standard hydrogen electrode, SHE) and thus the weakest affinity for electrons and therefore strongest tendency to donate them. It is supposed that under polarization conditions reduced NADH is also able to donate electrons to molecules, which act as electron/metabolite shuttles, pass through the porous cell wall, and transfer them to the anode.

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Krebs Cycle Takes Place in What Part of the Cell

Source: https://www.sciencedirect.com/topics/engineering/krebs-cycle

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