6.1: Metabolism - Sugars

Glycolysis

Carbohydrates, whether synthesized by photosynthetic organisms, stored in cells as glycogen, or ingested by heterotrophs, must be broken down to obtain energy for the cell’s activities as well as to synthesize other molecules required by the cell. Starch and glycogen, polymers of glucose, are the main energy storage forms of carbohydrates in plants and animals, respectively. To use these sources of energy, cells must first break down the polymers to yield glucose. The glucose is then taken up by cells through transporters in cell membranes. The metabolism of glucose, as well as other six carbon sugars (hexoses) begins with the catabolic pathway called glycolysis. In this pathway, sugars are oxidized and broken down into pyruvate molecules. The corresponding anabolic pathway by which glucose is synthesized is termed gluconeogenesis. Neither glycolysis nor gluconeogenesis is a major oxidative/reductive process, with one step in each one involving loss/gain of electrons, but the product of glycolysis, pyruvate, can be completely oxidized to carbon dioxide (Figure 6.2). Indeed, without production of pyruvate from glucose in glycolysis, a major energy source for the cell would not be available.

Glucose is the most abundant hexose in nature and is traditionally used to illustrate the reactions of glycolysis, but fructose (in the form of fructose-6- phosphate) is also readily metabolized, while galactose can easily be converted into glucose for catabolism in the pathway as well. The end metabolic products of glycolysis are two molecules of ATP, two molecules of NADH and two molecules of pyruvate (Figure 6.3), which, in turn, can be oxidized further in the citric acid cycle.

Entry points for glycolysis

Glucose and fructose are the sugar ‘funnels’ serving as entry points to the glycolytic pathway. Other sugars must be converted to either of these forms to be metabolized in glycolysis. Some pathways, including the Calvin

Figure 6.2 - Metabolic fates of glucose Image by Aleia Kim Your cells may have a mounting crisis Should they not go through glyco-lye-sis No glucose energy releases Until it’s fractured into pieces

Figure 6.3 - Glycolysis and its Regulators Image by Ben Carson Cycle and the Pentose Phosphate Pathway (PPP) contain intermediates in common with glycolysis, so in that sense, almost any cellular sugar can be metabolized here.

Other pathways

Intermediates of glycolysis and gluconeogenesis that are common to other pathways include glucose-6-phosphate (PPP, glycogen metabolism), Fructose-6-phosphate (Calvin Cycle, PPP), Glyceraldehyde-3- phosphate (Calvin Cycle, PPP), dihydroxyacetone phosphate (PPP, glycerol metabolism, Calvin Cycle), 3- phosphoglycerate (Calvin Cycle, PPP), phosphoenolpyruvate (C4 plant metabolism, Calvin Cycle), and pyruvate (fermentation, acetyl-CoA genesis, amino acid metabolism). It is worth noting that glycerol from the breakdown of fat can readily be metabolized to dihydroxyacetone phosphate (DHAP) and thus enter the glycolysis pathway. It is the only part of a fat that is used in these pathways.

Reaction 1

Glucose gets a phosphate from ATP to make glucose-6-phosphate (G6P) in a reaction catalyzed by the enzyme hexokinase, a transferase enzyme.

Glucose + ATP ⇄ G6P + ADP + H+

Hexokinase is one of three regulated enzymes in glycolysis and is inhibited by one of the products of its action - G6P. Hexokinase has flexibility in its substrate binding and is able to phosphorylate a variety of hexoses, including fructose, mannose, and galactose.

Why phosphorylate glucose?

Phosphorylation of glucose serves two important purposes. First, the addition of a phosphate group to glucose effectively traps it in the cell, as G6P cannot diffuse across the lipid bilayer. Second, the reaction decreases the concentration of free glucose, favoring additional import of the molecule. G6P is a substrate for the pentose phosphate pathway and can also be converted to glucose-1-phosphate (G1P) for use in glycogen synthesis and galactose metabolism (Figure 6.5).

It is worth noting that the liver has an enzyme like hexokinase called glucokinase, which Figure 6.4 - Reaction #1 - Phosphorylation of glucose - catalyzed by hexokinase has a much higher Km (lower affinity) for glucose. This is important, because the liver is a site of glucose synthesis (gluconeogenesis) where cellular concentrations of glucose can be relatively high. With a lower affinity glucose phosphorylating enzyme, glucose is not converted to G6P unless glucose concentrations get high, so the liver is able to release the glucose it makes into the bloodstream for the rest of the body to use.

Reaction 2

Next, G6P is converted to fructose-6-phosphate (F6P), in a reaction catalyzed by the enzyme

$\ce{G6P ⇄ F6P}$

The reaction has a low ΔG°’ , so it is readily favorable in either direction with Figure 6.6 - Mechanism of conversion of G6P to F6P in reaction #2 Figure 6.5 - The centrality of glucose-6-phosphate in metabolism Image by Aleia Kim only slight changes in concentration of reactants.

Reaction 3

$ce{F6P + ATP ⇄ F1,6BP + ADP + H+}$

The second input of energy occurs when F6P gets another phosphate from ATP in a reaction catalyzed by the enzyme phosphofructokinase-1 (PFK-1 - another transferase) to make fructose-1,6- bisphosphate (F1,6BP). PFK-1 is a very important enzyme regulating glycolysis, with several allosteric activators and inhibitors (see HERE).

Like the hexokinase reaction the energy from ATP is needed to make the reaction energetically favorable. PFK-1 is the most important regulatory enzyme in the pathway and this reaction is the ratelimiting step. It is also one of three essentially irreversible reactions in glycolysis.

A variant enzyme found in plants and some bacterial uses pyrophosphate rather than ATP as the energy source and due to the lower energy input from hydrolysis of the pyrophosphate, that reaction is reversible.

Reaction 4

$\ce{F1,6BP ⇄ D-GLYAL3P + DHAP}$

With the glycolysis pump thus primed, the pathway proceeds to split the F1,6BP into two 3-carbon intermediates. This reaction catalyzed by the lyase known as aldolase is energetically a “hump” to overcome in the glycolysis direction (∆G°’ = +24 kJ/mol Figure 6.7 - Reaction #3 - Conversion of F6P to F1,6BP by PFK Wikipedia Figure 6.8 - Reaction #4 - Breakdown of F1,6BP into GLYAL3P (left) and DHAP (right) by aldolase °K) so to get over the energy hump, cells must increase the concentration the reactant (F1,6BP) and decrease the concentration of the products, which are D-glyceraldehyde- 3-phosphate (D-GLYAL3P) and dihydroxyacetone phosphate (DHAP).

A novel scheme facilitates decreasing concentration of the products (see below). Aldolases cut the ketose ring by two different mechanisms and these enzymes are grouped as Class I (in animals and plants) and Class II (in fungi and bacteria).

Reaction 5

$\ce{DHAP ⇄ D-GLYAL3P}$

In the next step, DHAP is converted to DGLYAL3P in a reaction catalyzed by the enzyme triosephosphate isomerase. At this point, the six carbon glucose molecule has been broken down to two units of three carbons each - D-GLYAL3P. From this point forward each reaction of glycolysis contains two of each molecule. Reaction #5 is fairly readily reversible in cells.

The enzyme is of note because it is one example of a “perfect enzyme.” Enzymes in this category have very high ratios of Kcat/Km that approach a theoretical maximum limited only by the diffusion of substrate into the active site of the enzyme. The apparent reason for the enzyme evolving in this way is that the mechanism of the reaction produces an unstable, toxic intermediate (Figure 6.9). With the reaction proceeding as rapidly as it does, there is less chance of the intermediate escaping and causing damage in the cell.

Reaction 6

$\ce{D-GLYAL3P + NAD+ + Pi D-1,3BPG + NADH + H+}$

Figure 6.9 - Reaction #5 - Triose phosphate isomerase with unstable, toxic intermediate (methyl glyoxal) Image by Ben Carson

In this reaction, D-GLYAL3P is oxidized in the only oxidation step of glycolysis catalyzed by the enzyme glyceraldehyde-3- phosphate dehydrogenase, an oxidoreductase. The aldehyde in this reaction is oxidized, then linked to a phosphate to make an ester - D-1,3-bisphospho-glycerate (D- 1,3BPG). Electrons from the oxidation are donated to NAD+, creating NADH.

NAD+ is a critical constituent in this reaction and is the reason that cells need a fermentation option at the end of the pathway (see below).

Note here that ATP energy was not required to put the phosphate onto the oxidized D-GLYAL3P. The reason for this is because the energy provided by the oxidation reaction is sufficient for adding the phosphate.

Reaction 7

$\ce{D-1,3BPG + ADP ⇄ 3PG + ATP}$

The two phosphates in the tiny 1,3BPG molecule repel each other and give the molecule high potential energy. This energy is utilized by the enzyme phosphoglycerate kinase (another transferase) to phosphorylate ADP and make ATP, as well as the product, 3-phosphoglycerate (3-PG). This is an example of a substrate-level phosphorylation. Such mechanisms for making ATP require an intermediate with a high enough energy to phosphorylate ADP to make ATP. Figure 6.10 - Reaction #6 - Oxidation of GLYAL3P, catalyzed by glyceraldehyde-3-phosphate dehydrogenase Figure 6.11 - Reaction #7 - Substrate-level Phosphorylation by 1,3-BPG

Though there are a few substrate level phosphorylations in cells (including another one at the end of glycolysis), the vast major of ATP is made by oxidative phosphorylation in the mitochondria (in animals). In addition to oxidative phosphorylation, plants also make ATP by photophosphorylation in their chloroplasts. Since there are two 1,3 BPGs produced for every glucose, the two ATPs produced in this reaction replenish the two ATPs used to start the cycle and the net ATP count at this point of the pathway is zero.

Reaction 8

$\ce{3-PG ⇄ 2-PG }$

Conversion of the 3-PG intermediate to 2-PG (2- phosphoglycerate) occurs by an important mechanism. An intermediate in this readily reversible reaction (catalyzed by phosphoglycerate mutase - a mutase enzyme) is 2,3-BPG. This intermediate, which is stable, is released with low frequency by the enzyme instead of being con- Figure 6.13 - Two routes to formation of 2,3-BPG Figure 6.14 - 2,3- Bisphosphoglycerate (2,3-BPG) Figure 6.12 - Reaction #8 - Conversion of 3-PG to 2-PG verted to 2-PG. 2,3BPG is important because it binds to hemoglobin and stimulates release of oxygen. The molecule can also be made from 1,3-BPG as a product of a reaction catalyzed by bisphophglycerate mutase (Figure 6.13). Cells which are metabolizing glucose rapidly release more 2,3-BPG and, as a result, get more oxygen, supporting their needs. Notably, cells which are metabolizing rapidly are using oxygen more rapidly and are more likely to be deficient in it.

Reaction 9

$\ce{2-PG ⇄ PEP + H2O}$

2-PG is converted by enolase (a lyase) to phosphoenolpyruvate (PEP) by removal of water, creating a very high energy intermediate. The reaction is readily reversible, but with PEP, the cell has one of its highest energy molecules and that is important for the next reaction.

Reaction 10

$\ce{PEP + ADP + H+ ⇄ PYR + ATP}$

Conversion of PEP to pyruvate by pyruvate kinase is the second substrate level phosphorylation of glycolysis, creating ATP. This reaction is what some refer to as the “Big Bang” of glycolysis because there is almost enough energy in PEP to stimulate production of a second ATP (ΔG°’ = 31.6 kJ/ mol), but it is not used. Consequently, this energy is lost as heat. If you wonder why you get hot when you exercise, the heat produced in the breakdown of glucose is a prime contributor and the pyruvate kinase reaction is a major source. Figure 6.16 - Reaction #10 - The big bang - PEP phosphorylates ADP with a lot of energy to spare Wikipedia Figure 6.15 - Reaction #9 - Enolase-catalyzed removal of water Wikipedia

Pyruvate kinase is the third and last enzyme of glycolysis that is regulated (see below). The primary reason this is the case is to be able to prevent this reaction from occurring when cells are making PEP while going through gluconeogenesis (see more HERE).

Catabolism of other sugars

Though glycolysis is a pathway focused on the metabolism of glucose and fructose, the fact that other sugars can be readily metabolized into glucose means that glycolysis can be used for extracting energy from them as well. Galactose is a good example. It is commonly produced in the produced in the body as a result of hydrolysis of lactose, catalyzed by the enzyme known as lactase (Figure 6.17). Deficiency of lactase is the cause of lactose intolerance.

Galactose begins preparation for entry into glycolysis by being converted to galactose-1- phosphate (catalyzed by galactokinase - Figure 6.18). Galactose-1-phosphate swaps with glucose-1-phosphate from UDP-glucose to make UDP-galactose (Figure 6.19). An epimerase converts UDPgalactose back to UDP-glucose and the cycle is complete. Each turn of the cycle thus takes in one galactose-1-phosphate and releases one glucose-1-phosphate.

Deficiency of galactose conversion enzymes results in accumulation of galactose (from breakdown of lactose). Excess galactose is converted to galactitol, a sugar alcohol. Galactitol in the human eye lens causes it to absorb water and this may be a factor in formation of cataracts.Figure 6.17 - Breakdown of lactose to glucose and galactose by lactase Image by Pehr Jacobson Figure 6.18 - Galactokinase Reaction Image by Penelope Irving Free fructose can also enter glycolysis by two mechanisms. First, it can be phosphorylated to fructose-6-phosphate by hexokinase. A more interesting alternate entry point is that shown in Figure 6.20. Phosphorylation of fructose by fructokinase produces fructose-1-phosphate and cleavage of that by fructose-1- phosphate aldolase yields DHAP and glyceraldehyde.

Phosphorylation of glyceraldehyde by triose kinase yields GLYAL3P. This alternative entry means for fructose may have important implications because DHAP and GLYAL3P are introduced into the glycolysis pathway while bypassing PFK-1 regulation. Some have proposed this may be important when considering metabolism of high fructose corn syrup, since it forces production of pyruvate, a precursor of acetyl-CoA, which is itself a precursor of fatty acids when ATP levels are high.

Mannose metabolism

Mannose can also be metabolized in glycolysis. In this case, it enters via fructose by the following two-step process - 1) phosphoryla- Figure 6.19 - Conversion of galactose-1-phosphate into glucose-6-phosphate Image by Aleia Kim tion by hexokinase to make mannose-6- phosphate followed by its conversion to fructose-6-phosphate, catalyzed by phosphomannoisomerase (Figure 6.21).

Glycerol metabolism

Glycerol is an important molecule for the synthesis of fats, glycerophospholipids, and other membrane lipids. Most commonly it is made into glycerol-3- phosphate (Figure 6.22) and the glycolysis/gluconeogenesis pathways are important both for producing the compound and for metabolizing it. The relevant intermediate in these pathways both for producing and for using glycerol-3-phosphate is DHAP. The enzyme glycerol-3-phosphate dehydrogenase reversibly converts glycerol-3- phosphate into DHAP (Figure 6.22).

This reaction, which is an oxidation, transfers electrons to NAD+ to produce NADH. In the reverse reaction, production of glycerol-3- phosphate from DHAP, of course, requires electrons from NADH for the reduction. Both glycolysis and gluconeogenesis are sources DHAP, meaning when the cell needs glycerol- 3-phosphate that it can use sugars (glucose, fructose, mannose, or galactose) as sources in glycolysis. For gluconeogenesis, sources include pyruvate, alanine and Figure 6.20 - Entry of fructose into glycolysis, bypassing PFK-1 Image by Penelope Irving Figure 6.21 - Entry of other sugars into glycolysis Image by Penelope Irving lactate (both can easily be made into pyruvate), oxaloacetate, aspartic acid (which can be made into oxaloacetate by transamination), and others. All of the intermediates of the citric acid cycle (and glyoxylate cycle) can be converted ultimately to oxaloacetate, which is a gluconeogenesis intermediate, as well.

It is worth noting that animals are unable to use fatty acids as materials for gluconeogenesis in net amounts, but they can, in fact, use glycerol in both glycolysis and gluconeogenesis. It is the only part of the fat molecule that can be so used.

Pyruvate metabolism

As noted, pyruvate produced in glycolysis can be oxidized to acetyl-CoA, which is itself oxidized in the citric acid cycle to carbon dioxide. That is not the only metabolic fate of pyruvate, though (Figure 6.23).

Pyruvate is a “starting” point for gluconeogenesis, being converted to oxaloacetate in the mitochondrion in the first step. Pyruvate in animals can also be reduced to lactate by adding electrons from NADH (Figure 6.24). This reaction produces NAD+ and is critical for generating the latter molecule to keep the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis (reaction #6) going under conditions when there is no oxygen.

This is because oxygen is necessary for the electron transport system (ETS) to operate and it performs the important function of converting NADH back to NAD+. When the ETS is running, NADH donates electrons to Complex I and is oxidized to NAD+ in the process, generating the intermediate needed for oxidizing GLYAL-3P. In the absence of oxygen, however, NADH cannot be converted to Figure 6.22 - Reactions in glycerol metabolism Image by Penelope Irving NAD+ by the ETS, so an alternative means of making NAD+ is necessary for keeping glycolysis running under low oxygen conditions (fermentation).

Bacteria and yeast generate NAD+ under oxygen deprived conditions by doing fermentation in a different way (Figure 6.25). They use NADH-requiring reactions that regenerate NAD+ while producing ethanol from pyruvate instead of making lactate. Thus, fermentation of pyruvate is essential to keep glycolysis operating when oxygen is limiting. It is also for these reasons that brewing of beer (using yeast) involves depletion of oxygen and muscles low in oxygen produce lactic acid (animals).

Pyruvate is a precursor of alanine which can be easily synthesized by transfer of a nitrogen from an amine donor, such as glutamic acid. Pyruvate can also be converted into oxaloacetate by carboxylation in the process of gluconeogenesis (see below).

The enzymes involved in pyruvate metabolism include pyruvate dehydrogenase (makes acetyl-CoA), lactate dehydrogenase (makes lactate), transaminases (make alanine), pyruvate carboxylase (makes ox- Figure 6.23 - Pyruvate metabolism. When oxygen is absent, pyruvate is converted to lactate (animals) or ethanol (bacteria and yeast). When oxygen is present, pyruvate is converted to acetyl-CoA. Not shown - Pyruvate transamination to alanine or carboxylation to form oxaloacetate. aloacetate), and pyruvate decarboxylase (a part of pyruvate dehydrogenase that makes acetaldehyde in bacteria and yeast).

Catalytic action and regulation of the pyruvate dehydrogenase complex is discussed in the section on the citric acid cycle (HERE).

Gluconeogenesis

The anabolic counterpart to glycolysis is gluconeogenesis (Figure 6.26), which occurs mostly in the cells of the liver and kidney and virtually no other cells in the body. In seven of the eleven reactions of gluconeogenesis (starting from pyruvate), the same enzymes are used as in glycolysis, but the reaction directions are reversed. Notably, the ∆G values of these reactions in the cell are typically near zero, meaning their direction can be readily controlled by changing substrate and product concentrations by small amounts.

The three regulated enzymes of glycolysis all catalyze reactions whose cellular ∆G values are not close to zero, making manipulation of reaction direction for their reac- Figure 6.24 - Formation of lactate in animal fermentation produces NAD+ for G3PDH Image by Ben Carson Figure 6.25 - Formation of ethanol in microbial fermentation produces NAD+ for G3PDH Image by Ben Carson tions non-trivial. Consequently, cells employ “work-around” reactions catalyzed by four different enzymes to favor gluconeogenesis, when appropriate.

Bypassing pyruvate kinase

Two of the enzymes (pyruvate carboxylase and PEP carboxykinase - PEPCK) catalyze reactions that bypass pyruvate kinase. F1,6BPase bypasses PFK-1 and G6Pase bypasses hexokinase. Notably, pyruvate carboxylase and G6Pase are found in the mitochondria and endoplasmic reticulum, respectively, whereas the other two are found in the cytoplasm along with all of the enzymes of glycolysis.

Biotin An important coenzyme used by pyruvate carboxylase is biotin (Figure 6.27). Biotin is commonly used by carboxylases to carry CO2 to incorporate into the substrate.

Also known as vitamin H, biotin is a water soluble B vitamin (B7) needed for many metabolic processes, including fatty acid synthesis, gluconeogenesis, and amino acid metabolism. Deficiency of the vitamin is rare, since it is readily produced by gut Gluconeogenesis and glycolysis. Only the enzymes differing in gluconeogenesis are shown Image by Aleia Kim teria. There are many claims of advantages of taking biotin supplements, but there is no strong indication of benefits in most cases. Deficiencies are associated with inborn genetic errors, alcoholism, burn patients, and people who have had a gastrectomy. Some pregnant and lactating women may have reduced levels due to increased biotin catabolism.

Reciprocal regulation

All of the enzymes of glycolysis and nine of the eleven enzymes of gluconeogenesis are all in the cytoplasm, necessitating a coordinated means of controlling them. Cells generally need to minimize the extent to which paired anabolic and catabolic pathways are occurring simultaneously, lest they produce a futile cycle, resulting in wasted energy with no tangible product except heat. The mechanisms of controlling these pathways have opposite effects on catabolic and anabolic processes. This method of control is called reciprocal regulation (see above).

Reciprocal regulation is a coordinated means of simultaneously controlling metabolic pathways that do opposite things. In reciprocal regulation, a single molecule (allosteric regulation) or a single covalent modification (phosphorylation/dephosphorylation,

Allosteric Regulation of Glycolysis & Gluconeogenesis

Reciprocal Regulation

AMP - Activates PFK-1, Inhibits F1,6BPase

F2,6BP - Activates PFK-1, Inhibits F1,6BPase

Citrate - Activates PFK-1, Inhibits F1,6BPase

Glycolysis Only

ATP - Inhibits PFK-1 and Pyruvate Kinase

Alanine - Inhibits Pyruvate Kinase

Gluconeogenesis Only

ADP - Inhibits Pyruvate Carboxylase and PEPCK

Acetyl-CoA - Activates Pyruvate Carboxylase

Figure 6.27 - Biotin carrying carbon dioxide (red) Wikipedia for example) has opposite effects on the different pathways.

Reciprocal allosteric effects For example, in glycolysis, the enzyme known as phosphofructokinase (PFK-1) is allosterically activated by AMP and a molecule known as F2,6BP (Figure 6.28). The corresponding enzyme from gluconeogenesis catalyzing a reversal of the glycolysis reaction is known as F1,6BPase. F1,6BPase is inhibited by both AMP and F2,6BP.

Reciprocal covalent effects

In glycogen metabolism, the enzymes phosphorylase kinase and glycogen phosphorylase catalyze reactions important for the breakdown of glycogen. The enzyme glycogen synthase catalyzes the synthesis of glyco- Directional velocity Inverts with reciprocity If glycolysis is flowing Glucose synthesis awaits But when the latter is a-going Sugar breakdown then abates Figure 6.28 - Regulation of glycolysis (orange path) and gluconeogenesis (black path) Image by Aleia Kim gen. Each of these enzymes is, at least partly, regulated by attachment and removal of phosphate.

Phosphorylation of phosphorylase kinase and glycogen phosphorylase has the effect of making them more active, whereas phosphorylation of glycogen synthase makes it less active. Conversely, dephosphorylation has the reverse effects on these enzymes - phosphorylase kinase and glycogen phosphorylase become less active and glycogen synthase becomes more active.

Simple and efficient

The advantage of reciprocal regulation schemes is that they are very efficient. It doesn’t take separate molecules or separate treatments to control two pathways simultaneously. Further, its simplicity ensures that when one pathway is turned on, the other is turned off.

This is especially important with catabolic/ anabolic regulation, because having both pathways going on simultaneously in a cell is not very productive, leading only to production of heat in a futile cycle. A simple futile cycle is shown on Figure 6.29. If unregulated, the cyclic pathway in the figure (shown in black) will make ATP in creating pyruvate from PEP and will use ATP to make oxaloacetate from pyruvate.

It will also use GTP to make PEP from oxaloacetate. Thus, each turn of the cycle will make one ATP, use one ATP and use one GTP for a net loss of energy. The process will start with pyruvate and end with pyruvate, so there is no net production of molecules. (see HERE for one physiological use of a futile cycle).

Specific gluconeogenesis controls

Besides reciprocal regulation, other mechanisms help control gluconeogenesis. First, PEPCK is controlled largely at the level of synthesis. Overexpression of PEPCK (stimulated by glucagon, glucocorticoid hormones, and cAMP and inhibited by insulin) produces symptoms of diabetes.

Pyruvate carboxylase is sequestered in the mitochondrion (one means of regulation) Figure 6.29 - A simple futile cycle - follow the black lines Image by Aleia Kim Interactive Learning Module HERE and is sensitive to acetyl-CoA, which is an allosteric activator. Acetyl-CoA concentrations increase as the citric acid cycle activity decreases. Glucose-6- phosphatase is present in low concentrations in many tissues, but is found most abundantly and importantly in the major gluconeogenic organs – the liver and kidney cortex.

Specific glycolysis controls

Control of glycolysis and gluconeogenesis is unusual for metabolic pathways, in that regulation occurs at multiple points. For glycolysis, this involves three enzymes:

1. Hexokinase (Glucose ⇄ G6P)

2. Phosphofructokinase-1 (F6P ⇄ F1,6BP)

3. Pyruvate kinase (PEP ⇄ Pyruvate).

Regulation of hexokinase is the simplest of these. The enzyme is unusual in being inhibited by its product, glucose-6-phosphate. This ensures when glycolysis is slowing down hexokinase is also slowing down to reduce feeding the pathway.

Pyruvate kinase

It might also seem odd that pyruvate kinase, the last enzyme in the pathway, is regulated (Figure 6.30), but the reason is simple. Pyruvate kinase catalyzes the most energetically rich reaction of glycolysis. The reaction is favored so strongly in the forward direction that cells must do a ‘two-step’ around it in the reverse direction when making glucose in the gluconeogenesis pathway. In other words, it takes two enzymes, two reactions, and two triphosphates (ATP and GTP) to go from one pyruvate back to one PEP in gluconeogenesis. When cells are needing to make glu- igure 6.30 - Regulation of pyruvate kinase For cells a glucose cycling’s cost Is energy in reams Four ATPs each time is lost From breaking/making schemes So use for metabolic heat To make it warm inside your feet Else it’s of no utility To practice such futility cose, they can’t be sidetracked by having the PEP they have made in gluconeogenesis be converted directly back to pyruvate by pyruvate kinase. Consequently, pyruvate kinase must be inhibited during gluconeogenesis or a futile cycle will occur and no glucose will be made.

Another interesting control mechanism called feedforward activation involves pyruvate kinase. Pyruvate kinase is activated allosterically by the glycolysis intermediate, F1,6BP. This molecule is a product of the PFK-1 reaction and a substrate for the aldolase reaction.

Reactions pulled

As noted above, the aldolase reaction is energetically unfavorable (high positive ∆G°’), thus allowing F1,6BP to accumulate. When this happens, some of the excess F1,6BP binds to pyruvate kinase, which activates and jump- Figure 6.31 - Regulation of Synthesis and Breakdown of F2,6BP Image by Penelope Irving starts the conversion of PEP to pyruvate. The resulting drop in PEP levels has the effect of “pulling” on the reactions preceding pyruvate kinase. As a consequence, the concentrations of GLYAL3P and DHAP fall, helping to pull the aldolase reaction forward.

PFK-1 regulation

PFK-1 has a complex regulation scheme. First, it is reciprocally regulated (relative to F1,6BPase) by three molecules. F2,6BP activates PFK-1 and inhibits F1,6BPase. PFK-1 is also allosterically activated by AMP, whereas F1,6BPase is inhibited. On the other hand, citrate inhibits PFK-1, but activates F1,6BPase.

PFK-1 is also inhibited by ATP and is exquisitely sensitive to proton concentration, easily losing activity when the pH drops only slightly. PFK- 1’s inhibition by ATP is noteworthy and odd at first glance because ATP is also a substrate whose increasing concentration should favor the reaction instead of inhibit it. The root of this conundrum is that PFK-1 has two ATP binding sites - one at an allosteric site that binds ATP relatively inefficiently and one that the active site that binds ATP with high affinity. Thus, only when ATP concentration is high is binding at the allosteric site favored and only then can ATP turn off the enzyme.

F2,6BP regulation

Regulation of PFK-1 by F2,6BP is simple at the PFK-1 level, but more complicated at the level of synthesis of F2,6BP. Despite having a name sounding like a glycolysis/ gluconeogenesis intermediate (F1,6BP), F2,6BP is not an intermediate in either pathway. Instead, it is made from fructose-6-phosphate and ATP by the enzyme known as phosphofructokinase-2 (PFK- 2 - Figure 6.31).

Cori cycle

With respect to energy, the liver and muscles act complementarily. The liver is the major or- Figure 6.32 - The Cori cycle Image by Aleia Kim gan in the body for the synthesis of glucose. Muscles are major users of glucose to make ATP. Actively exercising muscles use oxygen faster than the blood can deliver it. As a consequence, the muscles go anaerobic and produce lactate. This lactate is of no use to muscle cells, so they dump it into the blood. Lactate travels in the blood to the liver, which takes it up and reoxidizes it back to pyruvate, catalyzed by the enzyme lactate dehydrogenase (Figure 6.32).

Pyruvate in the liver is then converted to glucose by gluconeogenesis. The glucose thus made by the liver is dumped into the bloodstream where it is taken up by muscles and used for energy, completing the important intercellular pathway known as the Cori cycle.

Glucose alanine cycle

The glucose alanine cycle (also known as the Cahill Cycle), has been described as the amine equivalent of the Cori cycle (Figure 6.33). The Cori cycle, of course, exports lac- Figure 6.33 - Overlap between the Cori cycle and the glucose alanine cycle tate from muscles (when oxygen is limiting) to the liver via the bloodstream. The liver, in turn, converts lactate to glucose, which it ships back to the muscles via the bloodstream. The Cori Cycle is an essential source of glucose energy for muscles during periods of exercise when oxygen is used faster than it can be delivered.

In the glucose-alanine cycle, cells are generating toxic amines and must export them. This is accomplished by transaminating pyruvate (the product of glycolysis) to produce the amino acid alanine.

The glucose-alanine process requires the enzyme alanine aminotransferase, which is found in muscles, liver, and intestines. Alanine is exported in the process to the blood and picked up by the liver, which deaminates it to release the amine for synthesis of urea and excretion. The pyruvate left over after the transamination is a substrate for gluconeogenesis. Glucose produced in the liver is then exported to the blood for use by cells, thus completing the cycle.

Polysaccharide metabolism

Sugars are metabolized rapidly in the body and that is one of the primary reasons they are used. Managing levels of glucose in the body is very important - too much leads to complications related to diabetes and too little gives rise to hypoglycemia (low blood sugar). Sugars in the body are maintained by three processes - 1) diet; 2) synthesis (gluconeogenesis); and 3) storage. The storage forms of sugars are, of course, the polysaccharides and their metabolism is our next topic of discussion.

Amylose and amylopectin

The energy needs of a plant are much less dynamic than those of animals. Muscular contraction, nervous systems, and information processing in the brain require large amounts of quick energy. Because of this, the polysaccharides stored in plants are somewhat less complicated than those of animals. Plants store glucose for energy in the form of amylose (Figure 6.34 and see HERE) and amylopectin and for structural integrity in the form of cellulose (see HERE). These structures differ in that cellulose contains glucose units solely joined by β-1,4 bonds, whereas amylose has only α-1,4 bonds and amylopectin has α-1,4 and α-1,6 bonds. Figure 6.34 Amylose, a polymer of glucose in plants

Glycogen

Animals store glucose primarily in liver and muscle in the form of a compound related to amylopectin known as glycogen. The structural differences between glycogen and amylopectin are solely due to the frequency of the α-1,6 branches of glucoses. In glycogen they occur about every 10 residues instead of every 30-50, as in amylopectin (Figure 6.35).

Glycogen provides an additional source of glucose besides that produced via gluconeogenesis. Because glycogen contains so many glucoses, it acts like a battery backup for the body, providing a quick source of glucose when needed and providing a place to store excess glucose when glucose concentrations in the blood rise.

The branching of glycogen is an important feature of the molecule metabolically as well. Since glycogen is broken down from the "ends" of the molecule, more branches translate to more ends, and more glucose that can be released at once.

Just as in gluconeogenesis, the cell has a separate mechanism for glycogen synthesis that is distinct from glycogen breakdown. As noted previously, this allows the cell to separately control the reactions, avoiding futile cycles, and enabling a process to occur efficiently (synthesis of glycogen) that would not occur if Figure 6.35 - Glycogen Structure - α-1,4 links with α-1,6 branches every 7-10 residues it were simply the reversal of glycogen breakdown.

Glycogen breakdown

Breakdown of glycogen involves 1) release of glucose-1-phosphate (G1P), 2) rearranging the remaining glycogen (as necessary) to permit continued breakdown, and 3) conversion of G1P to G6P for further metabolism. G6P can be 1) used in glycolysis, 2) converted to glucose by gluconeogenesis, or 3) oxidized in the pentose phosphate pathway.

Glycogen phosphorylase (sometimes simply called phosphorylase) catalyzes breakdown of glycogen into glucose-1- Phosphate (G1P - Figure 6.36). The reaction that produces G1P from glycogen is a phosphorolysis, not a hydrolysis reaction. The distinction is that hydrolysis reactions use water to cleave bigger molecules into smaller ones, but phosphorolysis reactions use phosphate instead for the same purpose. Note that the phosphate is just that - it does NOT come from ATP. Since ATP is not used to put phosphate on G1P, the reaction saves the cell energy.

Glycogen debranching enzyme

Glycogen phosphorylase will only act on nonreducing ends of a glycogen chain that are at least 5 glucoses away from a branch point. A second enzyme, Glycogen Debranching Enzyme (GDE) (also called debranching enzyme), is therefore needed to convert α (1-6) branches to α (1-4) branches. GDE acts on glycogen branches that have reached their limit of phosphorylysis with glycogen phosphorylase. Figure 6.36 - Breaking of α-1,4 bonds of glycogen by glycogen phosphorylase Image by Aleia Kim Interactive Learning Module HERE

GDE acts to transfer a trisaccharide from an α-1,6 branch onto an adjacent α-1,4 branch, leaving a single glucose at the 1,6 branch. Note that the enzyme also catalyzes the hydrolysis of the remaining glucose at the 1,6 branch point (Figure 6.37). Thus, the breakdown products from glycogen are G1P and glucose (mostly G1P). Glucose can, of course, be converted to Glucose-6-Phosphate (G6P) as the first step in glycolysis by either hexokinase or glucokinase.

G1P can be converted to G6P by action of an enzyme called phosphoglucomutase. This reaction is readily reversible, allowing G6P and G1P to be interconverted as the concentration of one or the other increases. This is important, because phosphoglucomutase is needed to form G1P for glycogen synthesis.

Regulation of glycogen metabolism

Regulation of glycogen metabolism is complex, occurring both allosterically and via hormone-receptor controlled events that result in protein phosphorylation or dephosphorylation. In order to avoid a futile cycle of glycogen synthesis and breakdown simultaneously, cells have evolved an elaborate set of controls that ensure only one pathway is primarily active at a time.

Regulation of glycogen metabolism is managed by the enzymes glycogen phosphorylase and glycogen synthase. Glycogen phosphorylase is regulated by both allosteric factors (ATP, G6P, AMP, and glucose) and by covalent modification (phosphorylation / dephosphorylation). Its regulation is consistent with the energy needs of the cell. High energy molecules (ATP, G6P, glucose) al- Figure 6.37 - Catalytic activity of debranching enzyme losterically inhibit glycogen phosphorylase, while the low energy molecule AMP allosterically activates it.

GPa/GPb allosteric regulation

Glycogen phosphorylase exists in two different covalent forms – one form with phosphate (called GPa here) and one form lacking phosphate (GPb here). GPb is converted to GPa by phosphorylation by an enzyme known as phosphorylase kinase. GPa and GPb can each exist in an 'R' state and a 'T' state (Figure 6.38). For both GPa and GPb, the R state is the more active form of the enzyme. GPa's negative allosteric effector (glucose) is usually not abundant in cells, so GPa does not flip into the T state often. There is no positive allosteric effector of GPa. When glucose is absent, GPa automatically flips into the R (more active) state (Figure 6.39). It is for this reason that people tend to think of GPa as being the more active covalent form of the enzyme.

GPb can convert from the GPb T state to the GPb R state by binding AMP. Unless a cell is low in energy, AMP concentration is low. Thus GPb is not converted Figure 6.38 - Glycogen phosphorylase regulation - covalent (horizontal) and allosteric (vertical) Image by Aleia Kim to the R state very often. This is why people think of the GPb form as less active than GPa. On the other hand, ATP and/or G6P are usually present at high enough concentration in cells that GPb is readily flipped into the T state (Figure 6.40).

GPa/GPb covalent regulation

The relative amounts of GPa and GPb largely govern the overall process of glycogen breakdown, since GPa tends to be active more often than GPb. It is i

Phosphorylase kinase itself has two covalent forms – phosphorylated (active) and dephosphorylated (inactive). It is phosphorylated by the enzyme Protein Kinase A (PKA - ). Another way to activate the enzyme is allosterically with calcium (Figure 6.41). Phosphory- Figure 6.39 - Allosteric regulation of GPa Image by Aleia Kim Figure 6.40 - Allosteric regulation of GPb Image by Aleia Kim lase kinase is dephosphorylated by phosphoprotein phosphatase, the same enzyme that removes phosphate from GPa.

PKA and cAMPcAMP

PKA is activated by cAMP, which is, in turn, produced by adenylate cyclase after activation by a G-protein (See HERE for overview). G-proteins are activated ultimately by binding of ligands to specific membrane receptors called 7-TM receptors, also known as Gprotein coupled receptors. These are discussed in greater detail HERE. Common ligands for 7-TM receptors include epinephrine (binds β- adrenergic receptor) and glucagon (binds glucagon receptor). Epinephrine exerts its greatest effects on muscle and glucagon works preferentially on the liver. Thus, epinephrine and glucagon can activate glycogen breakdown by stimulating synthesis of cAMP followed by the cascade of events described above.

Turning off glycogen breakdown

Turning off signals is as important, if not more so, than turning them on. Glycogen is a precious resource. If its breakdown is not controlled, a lot of energy used in its synthesis is wasted. The steps in the glycogen breakdown regulatory pathway can be reversed at every level. First, the ligand (epinephrine or glucagon) can leave the receptor, turning off the stimulus. Second, the G-proteins have an inherent GTPase activity. GTP, of course, is what activates Gproteins, so a GTPase activity converts the GTP it is carrying to GDP and the G-protein becomes inactive. Thus, G-proteins turn off Figure 6.41 - Activation of phosphorylase kinase Image by Aleia Kim their own activity. Interfering with their ability to convert GTP to GDP can have dire consequences, including cancer in some cases.

Third, cells have phosphodiesterase enzymes (inhibited by caffeine) for breaking down cAMP. cAMP is needed to activate PKA, so breaking it down stops PKA from activating phosphorylase kinase. Fourth, the enzyme known as phosphoprotein phosphatase (also called PP1) plays a major role. It can remove phosphates from phosphorylase kinase (inactivating it) and form GPa, converting it to the less likely to be active GPb. Regulation of phosphoprotein phosphatase activity occurs at several levels. Two of these are shown in Figures 6.42 & 6.43.

In Figure 6.42, phosphoprotein phosphatase is shown being inactivated by phosphorylation of an inhibitor (called PI-1 - see below). This happens as a result of cascading actions from binding of epinephrine (or glucagon) to a cell’s β-adrenergic receptor. Reversal of these actions occurs when insulin binds to the cell’s insulin receptor, resulting in activation of phosphoprotein phosphatase.

PI-1

The inhibitor PI-1 can block activity of phosphpoprotein phosphatase only if it (PI-1) is phosphorylated. When PI-1 gets dephosphorylated, it no longer functions as an inhibitor, so phosphoprotein phosphatase be- Figure 6.42 - Inactivation of phosphoprotein phosphatase by protein kinase A via phosphorylation of PI-1 (Inhibitor) and the GM binding protein Image by Pehr Jacobson Interactive Learning Module HERE comes active. Now, here is the clincher - PI-1 gets phosphorylated by PKA (thus, when epinephrine or glucagon binds to a cell) and gets dephosphorylated when insulin binds to a cell.

Another regulatory mechanism

Another way to regulate phosphoprotein phosphatase in the liver involves GPa directly (Figure 6.43). In liver cells, phosphoprotein phosphatase is bound to a protein called GL. GL can also bind to GPa. As shown in the figure, if the three proteins are complexed together (top of figure), then PP1 (phosphoprotein phosphatase) is inactive. When glucose is present (such as when the liver has made too much glucose), then the free glucose binds to the GPa and causes GPa to be released from the GL.

This has the effect of activating phosphoprotein phosphatase, which begins dephosphorylating enzymes. As shown in the figure, two such enzymes are GPa (making GPb) and glycogen synthase b, making glycogen synthase a. These dephosphorylations have opposite effects on the two enzymes, making GPb, which is less active and glycogen synthase a, which is much more active.

Glycogen synthesis

The anabolic pathway opposing glycogen breakdown is that of glycogen synthesis. Just Figure 6.43 - Regulation of phosphoprotein phosphatase (PP-1) activity by GPa Image by Penelope Irving as cells reciprocally regulate glycolysis and gluconeogenesis to prevent a futile cycle between these pathways, so too do cells use reciprocal schemes to regulate glycogen breakdown and synthesis.

Synthesis of glycogen starts with G1P, which is converted to an 'activated' intermediate, UDPglucose. This activated intermediate is what 'adds' the glucose to the growing glycogen chain in a reaction catalyzed by the enzyme known as glycogen synthase (Figure 6.44). Once the glucose is added to glycogen, the glycogen molecule may need to have branches inserted in it by the enzyme known as branching enzyme (Figure 6.45).

Steps

Let us first consider the steps in glycogen synthesis. 1) Glycogen synthesis from glucose involves phosphorylation to form G6P, and isomerization to form G1P (using phospho- igure 6.45 - Branch formation in glycogen by branching enzyme Image by Penelope Irving Figure 6.44 - Catalytic activity of glycogen synthase Image by Penelope Irving glucomutase, common to glycogen breakdown). G1P is reacted with UTP to form UDP-glucose in a reaction catalyzed by UDP-glucose pyrophosphorylase. Glycogen synthase catalyzes synthesis of glycogen by joining carbon #1 of the UDP-derived glucose onto the carbon #4 of the non-reducing end of a glycogen chain, to form the familiar α(1,4) glycogen links. Another product of the reaction is UDP.

“Primer” requirements

It is also worth noting, in passing, that glycogen synthase will only add glucose units from UDP-Glucose onto a preexisting glycogen chain that has at least four glucose residues. Linkage of the first few glucose units to form the minimal "primer" needed for glycogen synthase recognition is catalyzed by a protein called glycogenin, which attaches to the first glucose and catalyzes linkage of the first eight glucoses by α(1,4) bonds. 3) The characteristic α(1,6) branches of glycogen are the products of the enzyme known as branching enzyme. Branching enzyme breaks α(1,4) chains and carries the broken chain to the carbon #6 and forms an α(1,6) linkage (Figure 6.45).

Regulation of glycogen synthesis

The regulation of glycogen biosynthesis is reciprocal to that of glycogen breakdown. It also has a cascading covalent modification system similar to the glycogen breakdown system described above. In fact, part of the system is identical to glycogen breakdown. Epinephrine or glucagon signaling stimulates adenylate cyclase to make cAMP, which activates PKA. Figure 6.46 - Reciprocal regulation by the phosphorylation cascade - glycogen breakdown activated / glycogen synthesis inhibited Image by Penelope Irving

Effect of phosphorylation

In glycogen synthesis, protein kinase A phosphorylates the active form of glycogen synthase (GSa), and converts it into the usually inactive b form (called GSb).

Note the conventions for glycogen synthase and glycogen phosphorylase. For both enzymes, the more active forms are called the 'a' forms (GPa and GSa) and the less active forms are called the 'b' forms (GPb and GSb). The major difference, however, is that GPa has a phosphate, but GSa does not and GPb has no phosphate, but GSb does.

Thus phosphorylation and dephosphorylation have opposite effects on the enzymes of glycogen metabolism (Figure 6.46). This is the hallmark of reciprocal regulation. It is of note that the less active glycogen synthase form, GSb, can be activated by G6P. Recall that G6P had the exactly opposite effect on GPb.

Glycogen synthase, glycogen phosphorylase (and phosphorylase kinase) can all be dephosphorylated by the same enzyme - phosphoprotein phosphatase - and it is activated when insulin binds to its receptor in the cell membrane.

Big picture

In the big picture, binding of epinephrine or glucagon to appropriate cell receptors stimulates a phosphorylation cascade which simultaneously activates breakdown of glycogen by glycogen phosphorylase and inhibits synthesis of glycogen by glycogen synthase. Epinephrine, is also known as adrenalin, and the properties that adrenalin gives arise from a large temporary increase of blood glucose, which powers muscles.

On the other hand, insulin stimulates dephosphorylation by activating phosphoprotein phosphatase. Dephosphorylation reduces action of glycogen phosphorylase (less glycogen breakdown) and activates glycogen synthase (starts glycogen synthesis). Our bodies make glycogen when blood glucose levels rise. Since high blood glucose levels are harmful, insulin stimulates cells to take up glucose. In the liver and in muscle cells, the uptaken glucose is made into glycogen. Figure 6.47 - Cotton - the purest natural form of cellulose Wikipedia Interactive Learning Module HERE

Cellulose synthesis

Cellulose is synthesized as a result of catalysis by cellulose synthase. Like glycogen synthesis it requires an activated intermediate to add glucose residues and there are two possible ones - GDP-glucose and UDPglucose, depending on which cellulose synthase is involved. In plants, cellulose provides support to cell walls.

The reaction catalyzed is shown next where Cellulosen = a polymer of [(1→4)-β-Dglucosyl] n units long.

The GDP-glucose reaction is the same except with substitution of GDP-glucose for UDP-Figure 6.48 - The Pentose Phosphate Pathway - Enzymes - 1 = G6P dehydrogenase / 2 = 6-Phosphogluconolactonase / 3 = 6-PG dehydrogenase / 4a = Ribose 5- phosphate isomerase / 4b = Ribulose 5-phosphate 3-epimerase / 5,7 = Transketolase / 6 = Transaldolase UDP-glucose + Cellulosen UDP + Cellulosen+1 glucose. UDP-glucose for the reaction is obtained by catalysis of sucrose synthase. The enzyme is named for the reverse reaction.

Pentose phosphate pathway

The pentose phosphate pathway (PPP - also called the hexose monophosphate shunt) is an oxidative pathway involving sugars that is sometimes described as a parallel pathway to glycolysis. It is, in fact, a pathway with multiple inputs and outputs (Figure 6.48). PPP is also a major source of NADPH for biosynthetic reactions and can provide ribose-5-phosphate for nucleotide synthesis.

Though when drawn out, the pathway’s “starting point” is often shown as glucose-6-phosphate (G6P), in fact there are multiple entry points including other glycolysis intermediates, such as fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (GLYAL-3-P), as well as less common sugar compounds with 4,5, and 7 carbons.

The multiple entry points and multiple outputs gives the cell tremendous flexibility to meet its needs by allowing it to use a variety of materials to make any of these products.

Oxidation #1

Beginning with G6P, PPP proceeds through its oxidative phase as follows:

The enzyme catalyzing the reaction is G6P dehydrogenase. It is the rate limiting step of the pathway and the enzyme is inhibited both by NADPH and acetyl-CoA. NADPH is important for anabolic pathways, such as fatty acid synthesis and also for maintaining glutathione in a reduced state. The latter is important in protection against damage from reactive oxygen species.

Deficiency of the G6P dehydrogenase enzyme is not rare, leading to acute hemolytic anemia, due to reduced NADPH concentration, and a reduced ability of the cell to disarm reactive oxygen species with glutathione. Reduced activity of the enzyme appears to have a protective effect against malarial infection, likely due to the increased fragility of the red blood cell membrane, which is then unable to sustain an infection by the parasite. Hydrolysis Reaction #2 is a hydrolysis and it is catalyzed by

Hydrolysis

Reaction #2 is a hydrolysis and it is catalyzed by 6-phosphogluconolactonase. The reac- Sucrose + UDP UDP-glucose + Fructose G6P + NADP+ 6-Phosphoglucono-δ-lactone + NADPH tion converts the circular 6-phosphoglucono- δ-lactone into the linear 6- phosphogluconate (6-PG) in preparation for oxidation in the next step.

Decarboxylation

Reaction #3 is the only decarboxylation in the PPP and the last oxidative step. It is catalyzed by 6-phosphogluconate dehydrogenase.

Mutations disabling the protein made from this gene negatively impact red blood cells. At this point, the oxidative phase of PPP is complete and the remaining reactions involve molecular rearrangements. Ru5P has two possible fates and these are each described below.

Isomerization

Reaction 4a: The enzyme catalyzing this reversible reaction is Ru5P isomerase (top of next column). It is important because this is the way cells make R-5-P for nucleotide synthesis. The R-5-P can also be used in other PPP reactions shown elsewhere.

Epimerization

Reaction 4b (catalyzed by Ru-5-P epimerase) is another source of a pentose sugars and provides an important substrate for subsequent reactions.

Transketolase reactions

The other reactions don’t really have an order to them and whether they occur or not depends on cellular needs. The first enzyme, transketolase, is flexible in terms of its substrate/product combinations and is used not only in PPP, but also in the Calvin cycle of plants. It catalyzes the next two reactions

In the first reaction (above), two phosphorylated sugars of 5 carbons each are converted into one phosphorylated sugar of 3 carbons and one of 7 carbons. In the second (next page), a five carbon sugar phosphate and aRu-5-P Xylulose-5-phosphate (Xu-5-P) Xu-5-P + R-5-P GLYAL-3-P + Sedoheptulose-7-phosphate (S-7-P) 6-PG + NADP+ Ribulose-5-phosphate (Ru-5-P) + NADPH + CO2 6-Phosphoglucono-δ-lactone + H2O 6-phosphogluconate (6-PG) + H+ Ru-5-P Ribose-5-phosphate (R-5-P) four carbon sugar phosphate are rearranged into sugar phosphates with 3 and 6 carbons.

Glycolysis intermediates

In the reversible reactions of the pentose phosphate pathway, one can see how glycolysis intermediates can easily be rearranged and made into other sugars. Thus, GLYAL-3-P and F6P can be readily made into Ribose-5- phosphate for nucleotide synthesis.

Involvement of F6P in the pathway permits cells to continue making nucleotides (by making R-5-P) or tryptophan (by making E- 4-P) even if the oxidative reactions of PPP are inhibited.

The last reaction is catalyzed by the enzyme known as transaldolase.

TPP co-factor

Transketolase uses thiamine pyrophosphate (TPP) to catalyze reactions. TPP’s thiaFigure 6.49 - Intermediates of the pentose phosphate pathway Xu-5-P + Erythrose-4-phosphate (E-4-P) GLYAL-3-P + F6P GLYAL-3-P + S-7-P E-4-P + F6P zole ring’s nitrogen and sulfur atoms on either side of a carbon, allow it to donate a proton and act as an acid, thus forming a carbanion, which gets stabilized by the adjacent tetravalent nitrogen (Figures 6.50 & 6.51)).

The stabilized carbanion plays important roles in the reaction mechanism of enzymes, such as transketolase that use TPP as a cofactor. Commonly, the carbanion acts as a nucleophile that attacks the carbonyl carbon of the substrate. Such is the case with transketolase. Attack by the carbanion breaks the carbonyl bond on the substrate and covalently links it to the ionized carbon of TPP, thus allowing it to “carry” the carbonyl group to the other substrate for attachment. In this way, two carbons are moved from Xu- 5-P to E-4-P to make F6P (from E-4-P) and GLYAL-3-P (from Xu-5-P). Similarly, S-7-P and GLYAL-3-P are made from R-5-P and Xu-5-P, respectively.

Thiamines

Thiamines are a class of compounds involved in catalysis of important respiration-related The Pentose Phosphate Pathway by Kevin Ahern I need erythrose phosphate And don’t know what to do My cells are full of G-6-P And NADP too But I just hit upon a plan As simple as can be I’ll run reactions through the path That’s known as PPP In just two oxidations There’s ribulose-5P Which morphs to other pentoses Each one attached to P The next step it is simple Deserving of some praise The pentose carbons mix and match Thanks to transketolase Glyceraldehyde’s a product Sedoheptulose is too Each with a trailing phosphate But we are not quite through Now three plus seven is the same As adding six and four By swapping carbons back and forth There’s erythrose-P and more At last I’ve got the thing I need From carbons trading places I’m happy that my cells are full Of some transaldolases Figure 6.50 - Thiamine pyrophosphate reactions in the citric acid cycle, pyruvate metabolism, the pentose phosphate pathway, and the Calvin cycle. Thiamine was the first water-soluble vitamin (B1) to be discovered via association with the peripheral nervous system disease known as Beriberi. Thiamine pyrophosphate (TPP) is an enzyme cofactor found in all living systems derived from thiamine by action of the enzyme thiamine diphosphokinase. TPP facilitates catalysis of several biochemical reactions essential for tissue respiration.

Deficiency of the vitamin is rare today, though people suffering from Crohn’s disease, anorexia, alcoholism or undergoing kidney dialysis may develop deficiencies. TPP is required for the oxidative decarboxylation of pyruvate to form acetyl-CoA and similar reactions. Transketolase, an important enzyme in the pentose phosphate pathway, also uses it as a coenzyme. Besides these reactions, TPP is also required for oxidative decarboxylation of α-keto acids like α-ketoglutarate and branched-chain α-keto acids arising from metabolism of valine, isoleucine, and leucine. Figure 6.51 - Mechanism of action of thiamine pyrophosphate (TPP) - 1) Carbanion formation; 2) Nucleophilic attack; 3) Covalent attachment of carbonyl; 4) Transfer to second group; 5) Release of product and regeneration of TPP

TPP acts in the pyruvate dehydrogenase complex to assist in decarboxylation of pyruvate and “carrying” the activated acetaldehyde molecule to its attachment (and subsequent oxidation) to lipoamide. Central to TPP’s function is the thiazolium ring, which stabilizes carbanion intermediates (through resonance) by acting as an electron sink (Figure 6.51). Such action facilitates breaking of carbon-carbon bonds such as occurs during decarboxylation of pyruvate to produce the activated acetaldehyde.

Thiamine deficiency

Thiamine is integral to respiration and is needed in every cell. Acute deficiency of thiamine leads to numerous problems - the best known condition is beriberi, whose symptoms include weight loss, weakness, swelling, neurological issues, and irregular heart rhythms. Figure 6.52 - The Calvin cycle - The resynthesis phase has multiple steps and is described below. Image by Aleia Kim

Causes of deficiency include poor nutrition, significant intake of foods containing the enzyme known as thiaminase, foods with compounds that counter thiamine action (tea, coffee), and chronic diseases, including diabetes, gastrointestinal diseases, persistent vomiting. People with severe alcoholism often are deficient in thiamine.

Calvin cycle

The Calvin cycle (Figure 6.52) is a metabolic pathway occurring exclusively in photosynthetic organisms. Commonly referred to as the “Dark Cycle” or the Light-Independent Cycle, the Calvin cycle does not actually occur in the dark. The cell/chloroplast simply is not directly using light energy to drive it.

Assimilation

It is in the Calvin cycle of photosynthesis that carbon dioxide is taken from the atmosphere and ultimately built into glucose (or other sugars). Reactions of the Calvin cycle take place in regions of the chloroplast known as the stroma, the fluid areas outside of the thylakoid membranes. The cycle can be broken into three phases

1) assimilation of CO2

2) reduction reactions

3) regeneration of the starting material, ribulose 1,5 bisphosphate (Ru1,5BP).

Though reduction of carbon dioxide to glucose ultimately requires electrons from twelve molecules of NADPH (and 18 ATPs), it is confusing because one reduction occurs 12 times (1,3 BPG to GLYAL-3P) to input the overall reduction necessary to make one glucose.

Carbon dioxide

Another reason students find the pathway confusing is because the carbon dioxide molecules are absorbed one at a time into six different molecules of Ru1,5BP. At no point are the six carbons ever together in the same molecule to make a single glucose.

Instead, six molecules of Ru1,5BP (30 carbons) gain six more carbons via carbon dioxide and then split into 12 molecules of 3- phosphoglycerate (36 carbons). The gain of six carbons allows two three carbon molecules to be produced in excess for each turn of the cycle. These two molecules molecules are then converted into glucose using the enzymes of gluconeogenesis. The other ten molecules of 3-PG are used to regenerate the six molecules of Ru1,5BP. Figure 6.53 - Rubisco, the most abundant enzyme on Earth

Cyclic pathway

Like the citric acid cycle, the Calvin cycle doesn’t really have a starting or ending point, but can we think of the first reaction as the fixation of carbon dioxide to Ru1,5BP. This reaction is catalyzed by the enzyme known as ribulose-1,5 bisphosphate carboxylase (RUBISCO - Figure 6.53). The resulting six carbon intermediate is unstable and is rapidly converted to two molecules of 3- phosphoglycerate.

As noted, if one starts with 6 molecules of Ru1,5BP and makes 12 molecules of 3-PG, the extra 6 carbons that are a part of the cycle can be shunted off as two three-carbon molecules of glyceraldehyde-3-phosphate (GLYAL3P) to gluconeogenesis, leaving behind 10 molecules to be reconverted into 6 moleFigure 6.54 - Resynthesis phase of the Calvin cycle - All paths lead to regenerating Ru1,5BP, which is the aim of the resynthesis phase. Glycolysis/gluconeogenesis intermediates shown in blue. Enzyme numbers explained in text. cules of Ru1,5BP. This occurs in what is called the resynthesis phase.

Resynthesis phase

The resynthesis phase (Figure 6.54) requires multiple steps, but only utilizes two enzymes unique to plants - sedoheptulose-1,7 bisphosphatase and phosphoribulokinase. RUBISCO is the third (and only other) enzyme of the pathway that is unique to plants.

All of the other enzymes of the pathway are common to plants and animals and include some found in the pentose phosphate pathway and gluconeogenesis. Enzymes shown as numbers in Figure 6.54 are as follows (enzymes unique to plants in green):

1 - Phosphoglycerate kinase

2 - G3PDH

3 - Triosephosphate Isomerase

4 - Aldolase

5 - Fructose 1,6 bisphosphatase

6 - Transketolase

7 - Phosphopentose Epimerase

8 - Phosphoribulokinase

9 - Sedoheptulose 1,7 bisphosphatase

10 - Phosphopentose Isomerase

Reactions

The resynthesis phase begins with conversion of the 3-PG molecules into GLYAL3P (there are actually 10 GLYAL3P molecules involved in resynthesis, as noted above, but we are omitting numbers to try to help students to see the bigger picture. Suffice it to say that there are sufficient quantities of all of the molecules to complete the reactions described). Some GLYAL3P is converted to DHAP by triose phosphate isomerase. Some DHAPs are converted (via gluconeogenesis) to F6P (one phosphate is lost for each F6P).

Two carbons from F6P are given to GLYAL3P to create E-4P and Xu-5P (reversal of PPP reaction). E- 4P combines with DHAP to form sedoheptulose-1,7 bisphosphate (S1,7BP). The phosphate at position #1 is Figure 6.55 - Use of CO2 (Calvin cycle) vs. O2 (photorespiration) by RUBISCO. Image by Pehr Jacobson cleaved by sedoheptulose-1,7 bisphosphatase to yield S-7-P. Transketolase (another PPP enzyme) catalyzes transfer to two carbons from S-7-P to GLYAL3P to yield Xu-5P and R5P.

Phosphopentose isomerase catalyzes conversion of R5P to Ru5P and phosphopentose epimerase similarly converts Xu-5P to Ru5P. Finally, phosphoribulokinase transfers a phosphate to Ru5P (from ATP) to yield Ru1,5BP.

Photorespiration

In the Calvin cycle of photosynthesis, the enzyme ribulose-1,5-bisphosphate carboxylase (RUBISCO) catalyzes the addition of carbon dioxide to ribulose-1,5- bisphosphate (Ru1,5BP) to create two molecules of 3-phosphoglycerate. Molecular oxygen (O2), however, competes with CO2 for this enzyme, so about 25% of the time, the molecule that gets added is not CO2, but rather O2 (Figure 6.55). When this happens, the following reaction occurs

This is the first step in the process known as photorespiration. The process of photorespiration is inefficient relative to the carboxylation of Ru1,5BP. Phosphoglycolate is converted to glyoxylate in the glyoxysome and then transamination of that yields glycine. Two glycines can combine in a complicated coupled set of reactions in the mitochondrion shown next. Figure 6.56 - Maize - a C4 plant Ru1,5BP + O2 Phosphoglycolate + 3-phosphoglycerate + 2H+ 2 Glycine + NAD+ + H2O Serine + CO2 + NH3 + NADH + H+

Deamination and reduction of serine yields pyruvate, which can be then be converted back to 3-phosphoglycerate. The end point of oxygenation of Ru1,5BP is the same as the carboxylation of Ru1,5BP reactions, but there are significant energy costs associated with it, making the process less efficient.

C4 plants

The Calvin cycle is the means by which plants assimilate carbon dioxide from the atmosphere, ultimately into glucose. Plants use two general strategies for doing so. The first is employed by plants called C3 plants (most plants) and it simply involves the pathway described above. They are called C3 plants because the first stable intermediate after absorbing carbon dioxide contains three carbons - 3-phosphoglycerate. Another class of plants, called C4 plants (Figure 6.56) employ a novel strategy for concentrating the Figure 6.57 - Assimilation of CO2 by C4 plants Image by Aleia Kim CO2 prior to assimilation. C4 plants are generally found in hot, dry environments where conditions would otherwise favor the wasteful photorespiration reactions of RUBISCO and loss of water.

Capture by PEP

In C4 plants, carbon dioxide is captured in special mesophyll cells first by phosphoenolpyruvate (PEP) to make oxaloacetate (contains four carbons and gives the C4 plants their name - Figure 6.57). The oxaloacetate is converted to malate and transported into bundle sheath cells where the carbon dioxide is released and captured by Ru1,5BP, as in C3 plants. The Calvin cycle proceeds from there. The advantage of the C4 plant scheme is that it allows concentration of carbon dioxide while minimizing loss of water and photorespiration.

Peptidoglycan synthesis

Bacterial cell walls contain a layer of protection known as the peptidoglycan layer. Assembly of the layer begins in the cytoplasm.

Steps in the process follow

1. Donation of an amine from glutamine to fructose-6- phosphate and isomerization to make glucosamine-6- phosphate.

2. Donation of an acetyl group from acetyl-CoA to make N-acetylglucosamine-6- phosphate

3. Isomerization of N-acetylglucosamine-6- phosphate makes N-acetylglucosamine-1- phosphate Figure 6.58 - Peptidoglycan layer in a bacterial outer cell wall Wikipedia

4. UTP combines with N-acetylglucosamine-1-phosphate to make UDP-N-acetyl-glucosamine-1- phosphate

6. A pentapeptide or tetrapeptide chain is attached to the UDP-Nacetylmuramic acid. The sequence varies a bit between species, but commonly is L-Ala - D-Glu - L-Lys - DAla - D-Ala

7. Dolichol phosphate replaces UMP on the UDP-N-acetylmuramic acid-pentapeptide.

8. UDP-N-acetyl-glucosamine donates a glucose to the Nacetylmuramic acid part of the Dolichol-PP-N-acetylmuramic acidpentapeptide

9. A pentapeptide chain of glycines (pentaglycine) is linked to lysine of the pentapeptide chain to create a Dolichol-PP-Nacetylmuramic acid-N-acetylglucosaminedecapeptide. The pentaglycine serves as cross links in the overall structure.

10. Dolichol-PP is removed to yield Nacetylmuramic acid-N-acetylglucosaminedecapeptide Figure 6.60 - Catalytic activity of DDtranspeptidase Wikipedia Figure 6.59 - Penicillin

11. This last group is added to the growing peptidoglycan network by joining the pentaglycine of one chain to the tetrapeptide/ pentapeptide of another.

The enzyme catalyzing the addition of the N-acetylmuramic acid-N-acetylglucosaminedecapeptide to the network in the last step is DD-transpeptidase. This is the cellular enzyme targeted by penicillin and its derivatives. One reason penicillin is so effective is because synthesis of a peptidoglycan cell wall for a single bacterium requires millions of cycles of reactions above. Even slowing down the process can have a major effect on bacterial growth. On the flip side, resistance to penicillin and derivatives arises as a result of mutations in one enzyme - the transpeptidase.

Metabolons

At this point, it is appropriate to bring up the concept of metabolons. Metabolons are cellular complexes containing multiple enzymes of a metabolic pathway that appear to be arranged so that the product of one enzymatic reaction is passed directly as substrate to the enzyme that catalyzes the next reaction in the metabolic pathway. The structural complexes are temporary and are held together by non-covalent forces.

Metabolons appear to offer advantages of reducing the amount of water needed to hydrate enzymes. Activity of enzymes in the complex is increased. Most of the basic metabolic pathways are thought to use metabolons. They include glycolysis, the citric acid cycle, nucleotide metabolism, glycogen synthesis, steroid synthesis, DNA synthesis, RNA synthesis, the urea cycle, and the process of electron transport.

Hypoxia

Hypoxia occurs when the body or a region of it has an insufficient oxygen supply. Varia- Figure 6.61 - Hypoxia inducible factor tions in arterial oxygen concentrations in normal physiology may lead to hypoxia, for example, during hypoventilation training or strenuous physical exercise. Generalized hypoxia may appear in healthy people when at high altitudes. Cancer cells, which may be undergoing faster respiration than surrounding tissues may also tend to be hypoxic. Hypoxia is an important consideration for sugar metabolism due to the ability of cells to change their sugar metabolism (fermentation) when these conditions exist.

The body’s response to hypoxia is to produce Hypoxia-Inducible Factors (HIFs), which are transcription factors that induce expression of genes to help cells adapt to the hypoxic conditions. Many of the genes activated by HIFs are enzymes of glycolysis and GLUTs (glucose transport proteins). The combination of these gene products allows cells to 1) import more glucose and 2) metabolize it more rapidly when it arrives. This is to be expected because anaerobic sugar metabolism is only about 1/15th as efficient as aerobic metabolism. Consequently, it requires much more sugar metabolism to keep the cancer cells alive. A recently discovered protein called cytoglobin is believed to help assist in hypoxia by facilitating transfer of oxygen from arteries to the brain.

Covalent modification

HIFs are regulated partly by an interesting covalent modification. When oxygen concentration is high, the enzyme prolyl hydroxylase will hydroxylate proline residues in HIFs. This stimulates the protein degradation system (proteasome) to degrade them. When oxygen concentration is low, the hydroxylation occurs to a much lower extent (or does not occur at all), reducing/stopping degradation of HIFs and allowing them to activate genes. In this way, the concentration of HIFs is kept high under low oxygen concentration (to activate HIF genes) and low under high oxygen concentrations (to stop synthesis of HIF genes).