CARBONHYDRATE METABOLISM1

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CHAPTER 2: CARBONHYDRATE METABOLISM- GLYCOLYTIC ENZYMES
INTERNATIONAL UNIVERSITY
SCHOOL OF BIOTECHNOLOGY
BIOCHEMISTRY
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Learning objectives
1. Review the carbonhydrates
2. Learn the names of the 10 enzymes of glycolysis.
3. Learn the structures of the intermediates in the glycolytic pathway.
4. Explore the structures of the glycolytic enzymes.
5. Understand the chemical mechanisms of the enzymes of glycolysis.

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Brief Content
1.The carbonhydrate
2.The glycolytic enzymes
2.1 Hexokinase
2.2 Phosphoglucose Isomerase
2.3 Phosphofructokinase
2.4 Aldolase
2.5 Triose phosphate Isomerase
2.6 Glyceraldehide-3- phosphate dehydrogenase
2.7 Phosphoglycerate kinase
2.8 Phosphoglycerate mutase
2.9 Enolase
2.10 Pyruvate kinase

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Detailed Content
1.The carbonhydrate
2.The glycolytic enzymes
2.1 Hexokinase
Structure
Catalytic mechanism
Active site details
2.2 Phosphoglucose Isomerase
Structure
Catalytic mechanism
Active site details
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Detailed Content
2.3 Phosphofructokinase
Structure
Catalytic mechanism
Active site details
2.4 Aldolase
Structure
Catalytic mechanism
Active site details
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Detailed Content
2.5 Triose phosphosphate Isomerase
Structure
Catalytic mechanism
Active site details
2.6 Glycealdehide-3- phosphate dehydrogenase
Structure
Catalytic mechanism
Active site details

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Detailed Content
2.7 Phosphoglycerate kinase
Structure
Catalytic mechanism
Active site details
2.8 Phosphoglycerate mutase
Structure
Catalytic mechanism
Active site details

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Detailed Content
2.9 Enolase
Structure
Catalytic mechanism
Active site details
2.10 Pyruvate kinase
Structure
Catalytic mechanism
Active site details
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1 CARBOHYDRATES:
Carbohydrates include both sugars and their polymers. The simplest carbohydrates are the monosaccharides, or single sugars, also known as simple sugars. Disaccharides are double sugars, consisting of two monosaccharides joined by condensation. The carbohydrates that are macromolecules are polysaccharides, polymers of many sugars.
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1.1. Sugars, the smallest carbohydrates, serve as fuel and carbon sources
- Monosaccharides (from the Greek monos, single, and sacchar, sugar) generally have molecular formulas that are some multiple of CH2O.
- Glucose (C6H12O6), the most common monosaccharide, is of central importance in the chemistry of life. In the structure of glucose, we can see the trademarks of a sugar:
- The molecule has a carbonyl group and multiple hydroxyl groups. Depending on the location of the carbonyl group, a sugar is either an aldose (aldehyde sugar) or a ketose (ketone sugar).
- Glucose, for example, is an aldose; fructose, a structural isomer of glucose, is a ketose. (Most names for sugars end in -ose. )

- Monosaccharides, particularly glucose, are major nutrients for cells. In the process known as cellular respiration, cells extract the energy stored in glucose molecules.
- Not only are simple sugar molecules a major fuel for cellular work, but their carbon skeletons serve as raw material for the synthesis of other types of small organic molecules, such as amino acids and fatty acids.
- Sugar molecules that are not immediately used in these ways are generally incorporated as monomers into disaccharides or polysaccharides.
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Fig 3.1. The structure and classification of some monosaccharides.
Sugars may be aldoses (aldehyde sugars) or ketoses (ketone sugars), depending on the location of the carbonyl group (pink). Sugars are also classified according to the length of their carbon skeletons. A third point of variation is the spatial arrangement around asymmetric carbons (compare, for example, the gray portions of glucose and galactose).
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Fig 3.2. Linear and ring forms of glucose
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- A disaccharide consists of two monosaccharides joined by a glycosidic linkage, a covalent bond formed between two monosaccharides by a dehydration reaction. For example, maltose is a disaccharide formed by the linking of two molecules of glucose.
- Also known as malt sugar, maltose is an ingredient for brewing beer. The most prevalent disaccharide is sucrose, which is table sugar. Its two monomers are glucose and fructose.
- Plants generally transport carbohydrates from leaves to roots and other nonphotosynthetic organs in the form of sucrose.
- Lactose, the sugar present in milk, is another disaccharide, consisting of a glucose molecule joined to a galactose molecule.
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Fig 3.3. 
Examples
of
disaccharide
synthesis
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1.2 Polysaccharides, the polymers of sugars,
have storage and structural roles
- Polysaccharides are macromolecules, polymers with a few hundred to a few thousand monosaccharides joined by glycosidic linkages.
- Some polysaccharides serve as storage material, hydrolyzed as needed to provide sugar for cells.
- Other polysaccharides serve as building material for structures that protect the cell or the whole organism.
- The architecture and function of a polysaccharide are determined by its sugar monomers and by the positions of its glycosidic linkages.
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- Starch, a storage polysaccharide of plants, is a polymer consisting entirely of glucose monomers. Most of these monomers are joined by 1-4 linkages (number 1 carbon to number 4 carbon), like the glucose units in maltose. The angle of these bonds makes the polymer helical.
- The simplest form of starch, amylose, is unbranched. Amylopectin, a more complex form of starch, is a branched polymer with 1-6 linkages at the branch points.
- Plants store starch as granules within cellular structures called plastids, including chloroplasts (see fig5-6a). By synthesizing starch, the plant can stockpile surplus glucose. Because glucose is a major cellular fuel, starch represents stored energy. The sugar can later be withdrawn from this carbohydrate bank by hydrolysis, which breaks the bonds between the glucose monomers.
- Animals store a polysaccharide called glycogen, a polymer of glucose that is like amylopectin but more extensively branched. Humans and other vertebrates store glycogen mainly in liver and muscle cells. Hydrolysis of glycogen in these cells releases glucose when the demand for sugar increases.
1.2.1 Storage polysaccharides
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Fig 3.4. Storage polysaccharides.
These examples, starch and glycogen, are composed entirely of glucose monomers, abbreviated here as hexagons. The polymer chains tend to spiral to form helices.
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- Organisms build strong materials from structural polysaccharides. For example, the polysaccharide called cellulose is a major component of the tough walls that enclose plant cells. On a global scale, plants produce almost 1011 (100 billion) tons of cellulose per year; it is the most abundant organic compound on Earth.
- Like starch, cellulose is a polymer of glucose, but the glycosidic linkages in these two polymers differ. The difference is based on the fact that there are actually two slightly different ring structures for glucose.
- When glucose forms a ring, the hydroxyl group attached to the number 1 carbon is locked into one of two alternative positions: either below or above the plane of the ring. These two ring forms for glucose are called alpha (a) and beta (b), respectively.
- In starch, all the glucose monomers are in the a configuration.
- In contrast, the glucose monomers of cellulose are all in the b configuration, making every other glucose monomer upside down with respect to the others.
1.2.2. Structural polysaccharides
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Fig 3.5. 
Starch and
cellulose structures
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- The differing glycosidic links in starch and cellulose give the two molecules distinct three-dimensional shapes. Whereas a starch molecule is mostly helical, a cellulose molecule is straight (and never branched), and its hydroxyl groups are free to hydrogen-bond with the hydroxyls of other cellulose molecules lying parallel to it.
- In plant cell walls, parallel cellulose molecules held together in this way are grouped into units called microfibrils. These cables are a strong building material for plants--as well as for humans, who use wood, which is rich in cellulose, for lumber.

- Enzymes that digest starch by hydrolyzing its a linkages are unable to hydrolyze the b linkages of cellulose. In fact, few organisms possess enzymes that can digest cellulose. Humans do not; the cellulose fibrils in our food pass through the digestive tract and are eliminated with the feces.
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Some microbes can digest cellulose, breaking it down to glucose monomers. Another important structural polysaccharide is chitin, the carbohydrate used by arthropods (insects, spiders, crustaceans, and related animals) to build their exoskeletons. An exoskeleton is a hard case that surrounds the soft parts of an animal. Pure chitin is leathery, but it becomes hardened when encrusted with calcium carbonate, a salt.

Chitin is also found in many fungi, which use this polysaccharide rather than cellulose as the building material for their cell walls.

- Chitin is similar to cellulose, except that the glucose monomer of chitin has a nitrogen-containing appendage:
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Fig 3.6. The arrangement of cellulose in plant cell walls.
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Fig 3.7. Chitin, a structural polysaccharide.
(a) Chitin forms the exoskeleton of arthropods. This cicada is molting, shedding its old exoskeleton and emerging in adult form.
(b) Chitin is used to make a strong and flexible surgical thread that decomposes after the wound or incision heals
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- The molecule known as ATP, short for adenosine triphosphate, is the central character in bioenergetics

- The triphosphate tail of ATP is the chemical equivalent of a loaded spring; the close packing of the three negatively charged phosphate groups is an unstable, energy-storing arrangement. The chemical "spring" tends to "relax" by losing the terminal phosphate

- The cell taps this energy source by using enzymes to transfer phosphate groups from ATP to other compounds, which are then said to be phosphorylated. Phosphorylation primes a molecule to undergo some kind of change that performs work, and the molecule loses its phosphate group in the process
ATP = ADP + Pi

- For example, a working muscle cell, for example, recycles its ATP at a rate of about 10 million molecules per second. To understand how cellular respiration regenerates ATP, we need to examine the fundamental chemical processes known as oxidation and reduction.
THE STRUCTURE OF ATP, NAD+
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Fig 3.8. The structure and hydrolysis of ATP.
The hydrolysis of ATP yields inorganic phosphate and ADP. In the cell, most hydroxyl groups of phosphates are ionized (--O-).
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Fig 3.9. NAD+ as an electron shuttle.
The full name for NAD+, nicotinamide adenine dinucleotide, describes its structure; the molecule consists of two nucleotides joined together. (Nicotinamide is a nitrogenous base, although not one that is present in DNA or RNA.) The enzymatic transfer of two electrons and one proton from some organic substrate to NAD+ reduces the NAD+ to NADH. Most of the electrons removed from food are transferred initially to NAD+.
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- Electrons lose very little of their potential energy when they are transferred from food to NAD+.
- Each NADH molecule formed during respiration represents stored energy that can be tapped to make ATP when the electrons complete their "fall" from NADH to oxygen

- How do electrons extracted from food and stored by NADH finally reach O2?
(1) The reaction between H2 and O2 to form H2O + gases = explosion + release of energy
(2) Cellular respiration also brings H2 and O2 together to form H2O, but there are two important differences. First, in cellular respiration, the H2 that reacts with O2 is derived from organic molecules. Second, respiration uses an electron transport chain to break the fall of electrons to O2 into several energy-releasing steps instead of one explosive reaction
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- The transport chain consists of a number of molecules, mostly proteins, built into the inner membrane of a mitochondrion.
- Electrons removed from food are shuttled by NADH to the "top" end of the chain. At the "bottom" end, O2 captures these electrons along with H2, forming water.

- Thus, electrons removed from food by NAD+ fall down the electron transport chain to a far more stable location in the electronegative O2 atom.

Food  NADH  electron transport chain 8n oxygen
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Fig 3.10. An introduction to electron transport chains.
(a) The uncontrolled exergonic reaction of H2 with O2 to form H2O releases a large amount of energy in the form of heat and light: an explosion.
(b) In cellular respiration, the same reaction occurs in stages: An electron transport chain breaks the "fall" of electrons in this reaction into a series of smaller steps and stores some of the released energy in a form that can be used to make ATP.
(The rest of the energy is released as heat.)
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THE PROCESS OF CELLULAR RESPIRATION
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Respiration involves glycolysis, the Krebs cycle,
and electron transport

- In a eukaryotic cell, glycolysis occurs outside the mitochondria in the cytosol.
The Krebs cycle and the electron transport chains are located inside the mitochondria.
- During glycolysis, each glucose molecule is broken down into two molecules of the compound pyruvate.
- The pyruvate crosses the double membrane of the mitochondrion to enter the matrix, where the Krebs cycle decomposes it to carbon dioxide.
- NADH or FADH2 transfers electrons from molecules undergoing glycolysis and the Krebs cycle to electron transport chains, which are built into the inner mitochondrial membrane.
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The electron transport chain converts the chemical energy to a form that can be used to drive oxidative phosphorylation, which accounts for most of the ATP generated by cellular respiration.
- A smaller amount of ATP is formed directly during glycolysis and the Krebs cycle by substrate-level phosphorylation.

(1) Glycolysis (color-coded teal throughout the chapter)
(2) The Krebs cycle (color-coded salmon)
(3) The electron transport chain and oxidative phosphorylation (color-coded violet)
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Fig.3.11: Overview of the cellular respiration
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Respiration is a cumulative function of 3 metabolic stages

(1) Glycolysis (color-coded teal throughout the chapter) - cytosol
Glycolysis, begins the degradation by breaking: glucose = two molecules pyruvate

(2) The Krebs cycle (color-coded salmon) - mitochondrial matrix
Decomposing a derivative of pyruvate to CO2


(3) The electron transport chain and oxidative phosphorylation (color-coded violet)
the electron transport chain accepts electrons from the breakdown products of the first two stages (usually via NADH) and passes these electrons from one molecule to another

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The energy released at each step of the chain is stored in a form the mitochondrion can use to make ATP.
- This mode of ATP synthesis is called oxidative phosphorylation because it is powered by the redox reactions that transfer electrons from food to O2.

- Oxidative phosphorylation accounts for almost 90% of the ATP generated by respiration
- A smaller amount of ATP is formed by substrate-level phosphorylation when an enzyme transfers a phosphate group from a substrate molecule to ADP. "Substrate molecule" here refers to an organic molecule generated during the catabolism of glucose.

cell respiration
glucose = CO2 + H2O + 38 molecules of ATP
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Fig 3.12. Substrate-level phosphorylation.
Some ATP is made by direct enzymatic transfer of a phosphate group from a substrate to ADP. The phosphate donor in this case is phosphoenolpyruvate (PEP), which is formed from the breakdown of sugar during glycolysis
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2. INTRODUCTION
Glycolysis is an almost universal pathway for extraction of the energy available from carbohydrates, shared among prokaryotes and eukaryotes, aerobes and anaerobes alike. In anaerobes, glycolysis is the only significant source of energy from carbohydrates. In aerobic organisms, considerably more energy can be harvested downstream from glycolysis in the citric acid cycle. Glycolysis produces energy in the form of ATP and NADH.

The glycolytic pathway consists of 10 enzyme-catalyzed steps. During glycolysis, glucose, a six-carbon carbohydrate, is oxidized to form two molecules of pyruvate, a three-carbon molecule. For each glucose molecule metabolized, the pathway produces two molecules of ATP and two molecules of NADH.
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2. INTRODUCTION
Glycolysis is not isolated from other metabolic pathways. Other molecules besides glucose can enter at a few points along the glycolytic pathway. For example, the product of glycogen breakdown glucose-6-phosphate, can enter the glycolytic pathway at the second step. Glyceraldehyde-3-phosphate, which is produced by photosynthesis, is also a glycolytic intermediate, so it can be directed from this anabolic pathway into glycolysis when energy is needed. Additionally, intermediates can be drawn out of the glycolytic pathway when energy levels are high, for use in biosynthetic pathways. For instance, during active energy production pyruvate, the product of glycolysis, enters the citric acid cycle, but when energy is not needed pyruvate serves as a substrate in amino acid synthesis.
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Fig 3.13: Reactions of Glycolysis
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Glycolysis harvests chemical energy by oxidizing glucose to pyruvate
- Glucose, a six-carbon sugar, is split into two three-carbon sugars. These smaller sugars are then oxidized and their remaining atoms rearranged to form two molecules of pyruvate. (Pyruvate is the ionized form of a three-carbon acid, pyruvic acid.)

- The pathway of glycolysis consists of ten steps, each catalyzed by a specific enzyme. We can divide these ten steps into two phases: The energy investment phase includes the first five steps, and the energy payoff phase includes the next five steps.
- During the energy investment phase, the cell actually spends ATP to phosphorylate the fuel molecules and NAD+ is reduced to NADH by oxidation of the food
glycolysis
glucose = 2 ATP + 2 NADH
2. GLYCOLYSIS
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Fig 3.14. The energy input and output of glycolysis
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Fig 3.15 . A closer look at glycolysis.
The orientation diagram at the right relates glycolysis to the whole process of respiration. Do not let the chemical detail in the main diagram block your view of glycolysis as a source of ATP and NADH.
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Fig 3.16. A closer look at glycolysis.
The orientation diagram at the right relates glycolysis to the whole process of respiration. Do not let the chemical detail in the main diagram block your view of glycolysis as a source of ATP and NADH.
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2. 1. Hexokinase
PHOSPHORYLATION OF GLUCOSE

The first glycolytic reaction attaches a phosphate group to glucose, to yield glucose-6-phosphate. This reaction is catalyzed by the enzyme hexokinase, shown here with its substrates bound in the active site. A kinase is an enzyme that catalyzes the transfer of a phosphoryl group to or from ATP. In this case, the phosphoryl group is transferred from ATP to glucose (thereby converting ATP to ADP).
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2. 1. Hexokinase
Structure

Hexokinase is a homodimer. Each subunit is made of two globular domains linked by an α helix. The subdomains are composed of two segments of β sheet that are protected from the solvent by α helices. The active sites are sandwiched between the β sheets. The active site has space to bind both glucose and ATP.
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3.17 The structure of hexokinase
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2.1. Hexokinase
Catalytic mechanism
The oxygen on carbon 6 of glucose performs α nucleophilic attack on the γ phosphate of ATP. The phosphate electron pair is donated to the anhydride oxygen bridging the β and γ phosphates of the ATP. Thus, the glucose obtains α phosphate from ATP.

Active site details
Glucose, the substrate of hexokinase, is cradled snugly in the enzyme`s active site. It forms hydrogen bonds with amino acid side chains and the protein backbone.
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3.18 Catalytic mechanism of hexokinase
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Fig. 3.19: Active site of hexokinase
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2. 2. Phosphoglucose Isomerase
ISOMERIZATION OF GLUCOSE-6-PHOSPHATE TO FRUCTOSE-6-PHOSPHATE

The second reaction is the isomerization of glucose-6-phosphate to fructose-6-phosphate by the enzyme phosphoglucose isomerase. Isomers have the same chemical formula (being composed of the same atoms), but with a different arrangement of bonds.
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2. 2. Phosphoglucose Isomerase
Structure

Phosphoglucose isomerase is a homodimer composed of two globular subunits that embrace each other with flanking a helical "arms." Each subunit is composed of one mixed and one parallel β sheet. These are separated by a central bundle of α helices. The active sites are located symmetrically at the interface between the two subunits near the flanking "arms."
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Fig 3.20: Structure of Phosphoglucose isomerase
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2.2 Phosphoglucose Isomerase
Catalytic mechanism

The mechanism of isomerization cannot proceed without first opening the ring of glucose-6-phosphate. An active site acid catalyzes the ring opening. A basic group then a abstracts the proton attached to carbon 2 on the glucose. This leads to double bond formation between carbon 1 and carbon 2 and an electron displacement at the carbonyl of carbon 1. In the subsequent step, a catalytic base abstracts a proton from the carbon 2 hydroxyl group, leaving an unpaired electron that proceeds to form a carbonyl bond with carbon 2. The remaining unpaired electron at carbon 1 then abstracts a proton from an active site acid and the isomerization is complete. Finally, ring closure produces the cyclic form of fructose-6-phosphate that is free to leave the active site.
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Fig 3.21: Catalytic mechanism of phosphoglucose Isomerase
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2.2 Phosphoglucose Isomerase
Active site details

The active site of phosphoglucose isomerase contains two amino acids believed to be directly involved in catalysis: a lysine acting as an acid and a conserved glutamate that functions as a base. Shown here is the active site with bound product fructose-6-phosphate.
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Fig. 3.22:Active site of phosphoglucose isomerase concluding lysine, histidine, glutamate
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2.3. Phospho- fructokinase
PHOSPHORYLATION OF FRUCTOSE-6-PHOSPHATE TO FRUCTOSE-1,6-BISPHOSPHATE

The third reaction in the glycolytic pathway is the phosphorylation of the fructose-6-phosphate by the enzyme phosphofructokinase (PFK) to produce fructose-1,6-bisphosphate. This step requires an ATP, and because of the highly favorable energetics of this irreversible reaction, it is known as the committed step of glycolysis and is highly regulated. Regulatory molecules include high-energy metabolic intermediates, such as phosphoenolpyruvate (PEP), that inhibit the activity of PFK, and low-energy intermediates, such as adenosine diphosphate (ADP), that activate PFK. Both activating and inhibitory effectors bind in the same binding pocket located between the subunits of each dimer of PFK.
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2.3. Phospho- fructokinase
Structure

PFK exists as a tetramer in solution. Four identical subunits associate to form the active form of PFK. Like hemoglobin, PFK is a dimer of dimers. Each subunit in the tetramer contains 319 amino acids that form two domains. The larger domain binds the substrate ATP, and the smaller domain binds the other substrate, fructose-6-phosphate. Each domain is a β barrel, consisting of a cylindrical β sheet surrounded by a helices.
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Fig. 3.23: Structure of Phospho- fructokinase
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2.3. Phospho- fructokinase
Catalytic mechanism

The catalytic mechanism of phosphofructokinase is nearly identical to the phosphorylation of glucose performed by hexokinase. The hydroxyl oxygen of carbon 1 performs a nucleophilic attack on the β phosphate of ATP. These electrons are donated to the anhydride oxygen bridging the β and γ phosphates of the ATP.
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Fig. 3.24: Catalytic mechanism of phosphofructokinase
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2. 3. Phospho- fructokinase
Active site details

The active site is located at the interface between subunits. In this crystal structure of PFK, ADP and glucose-6-phosphate are bound to the active site.
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Fig. 3. 25: Active site of Phospho- fructokinase including lysine and histidine
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2. 3. Phospho- fructokinase
Allosteric effector site

The allosteric effector site is located on the opposite side of each subunit from the active site, at the interface between subunits in the dimer. This structure has ADP bound at this site.
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Fig. 3.26: Allosteric effector site of Phospho- fructokinase
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2. 4. Aldolase
CLEAVAGE OF FRUCTOSE-1,6-BISPHOSPHATE INTO DIHYDROXYACETONE PHOSPHATE AND GLYCERALDEHYDE-3- PHOSPHATE

In the fourth reaction, the enzyme aldolase cleaves the hexose fructose-1,6-bisphosphate into two three-carbon molecules, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
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2. 4. Aldolase
Structure
Aldolase is a tetramer of identical subunits. Each subunit is composed of α β barrel surrounded by β helices. The active site is located at one end of the β barrel and can be identified by the bound product dihydroxyacetone phosphate.
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Fig. 3.27: Structure of aldolase
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2. 4. Aldolase
Catalytic mechanism

The mechanism of aldolase involves the formation of a Schiff base, a common covalent intermediate. In the first step, an active-site lysine donates an electron to the carbon 2 carbonyl of fructose-1,6-bisphosphate. Subsequent loss of water leads to the formation of the Schiff base. A tyrosine anion abstracts a proton from the carbon 4 hydroxyl group, leaving a carbonyl group. The displaced electrons at carbon 3 form an enolate intermediate with carbon 2. The displaced electron of the Schiff base now relocates to the nitrogen. At this point, glyceraldehyde-3-phosphate has been created and is free to diffuse out of the active site.
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2. 4. Aldolase
In subsequent steps, the Schiff base is reestablished, and the enolate intermediate is destroyed when the protonated tyrosine donates its proton to the displaced electron of carbon 3. Finally, a hydration step transforms the Schiff base back to a carbonyl, and the dihydroxyacetone phosphate product diffuses out of the active site.
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Fig. 3.28: Catalytic mechanism of aldolase
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2. 4. Aldolase
Active site details

Here you see the active site of aldolase complexed with glyceraldehyde-3-phosphate There is a histidine in the active site as well as several lysines that might be the Schiff-base forming residue.
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Fig. 3.29: Active site details of aldolase
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2. 5. Triose Phosphate Isomerase
ISOMERIZATION OF DIHYDROXYACETONE PHOSPHATE TO GLYCERALDEHYDE- 3-PHOSPHATE

The fifth reaction in the glycolytic pathway is the isomerization of dihydroxyacetone phosphate to glyceraldehyde-3-phosphate. Triose phosphate isomerase is an example of a "perfect" enzyme or an enzyme in which the rate of catalysis is limited only by the ability of the enzyme to encounter its substrate, rather than any chemical event.
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2. 5. Triose Phosphate Isomerase
Structure
Triose phosphate isomerase is a dimer made
from two globular subunits. Each subunit is composed of a central β barrel surrounded by α helices. In this respect, the secondary structure is similar to the previous enzyme in the pathway, aldolase.
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Fig. 3.30: Structure of Triose Phosphate Isomerase
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2.5. Triose Phosphate Isomerase
Catalytic mechanism

In the first step of the mechanism, an acid donates a proton to the carbonyl at carbon 2 of dihydroxyacetone phosphate. At the same time, a basic group abstracts a proton at carbon 1. This concerted action leads to the formation of an enediol intermediate. Subsequently, the deprotonated acid group accepts a proton from the hydroxyl group at carbon 1, as the protonated basic group donates its proton to carbon 2. This generates the glyceraldehyde-3-phosphate product. A glutamate and a histidine act as the general acids and bases for this isomerization reaction, and a lysine serves to stabilize the negative charge of the transition state intermediate.
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Fig. 3.31: Catalytic mechanism of Triose Phosphate Isomerase
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2.5. Triose Phosphate Isomerase

Active site details
The structure of triose phosphate isomerase can be seen here with glycerol-3-phosphate, an analog of glyceraldehyde-3-phosphate.
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Fig. 3.32: Active site details of Triose Phosphate Isomerase
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2. 6. Glyceraldehyde- 3-phosphate Dehydrogenase
OXIDATION AND PHOSPHORYLATION OF GLYCERALDEHYDE-3- PHOSPHATE

In the sixth reaction of glycolysis, glyceraldehyde-3-phosphate is both oxidized and phosphorylated to produce 1,3-bisphosphoglycerate. Unlike the kinases that catalyzed reactions 1 and 3, glyceraldehyde-3-phosphate dehydrogenase does not use ATP as a phosphoryl-group donor; it instead adds inorganic phosphate directly to the substrate. This reaction is also an oxidation-reduction reaction in which the aldehyde group of glyceraldehyde-3-phosphate is oxidized with the concomitant reduction of the cofactor NAD+ to NADH.
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2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase
Recent evidence supports a wide variety of cellular roles for this enzyme beyond its glycolytic function, including endocytosis, translational control, tRNA export from the nucleus, and DNA repair. Enzymes involved in more than one cellular process often serve as important regulators, connecting multiple enzymatic pathways.
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2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase
Structure

Glyceraldehyde-3-phosphate dehydrogenase is a tetramer. Each identical subunit is composed of two extended β sheets, both supported by three α helices.
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Fig. 3.33: Structure of Glyceraldehyde- 3-phosphate
Dehydrogenase
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2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase
Catalytic mechanism

In the first step, an active site sulfhydryl performs a nucleophilic attack on carbon 1 of glyceraldehyde-3-phosphate, forming a covalent thioester intermediate. The hydrogen on carbon 1 is donated to an NAD+ cofactor in the active site, forming NADH. An inorganic phosphate then performs a nucleophilic attack on carbon 1, displacing the sulfur as a sulfanion, which is quickly protonated by another enzyme group. 1,3-bisphosphoglycerate is now free to diffuse out of the active site.
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Fig. 3.34: Catalytic mechanism of Glyceraldehyde- 3-phosphate
Dehydrogenase
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2. 6. Glyceraldehyde- 3-phosphate
Dehydrogenase
Active site details

In the animation, you can see the active site of glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 3..35: Active site details of Glyceraldehyde- 3-phosphate
Dehydrogenase
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2. 7. Phosphoglycerate Kinase
DEPHOSPHORYLATION OF 1,3-BISPHOSPHOGLYCERATE TO FORM 3-PHOSPHOGLYCERATE

In the seventh reaction of the glycolytic pathway, phosphoglycerate kinase dephosphorylates 1,3-bisphosphoglycerate to form 3-phosphoglycerate. The free energy released in this reaction is used to drive the formation of ATP, as 1,3-bisphosphoglycerate donates its phosphoryl group to ADP. Note that phosphoglycerate kinase transfers a phosphoryl group to ADP to form ATP, whereas hexokinase and PFK catalyze phosphoryl group transfers from ATP.
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2. 7. Phosphoglycerate Kinase
Structure

Each subunit of phosphoglycerate kinase is composed of two subdomains made of a central β sheet surrounded by a bundle of α helices. The active site is divided between the two domains, shown here with the ADP bound to one domain and 1,2-bisphosphoglycerate, which is a substrate analog, bound to the other domain.
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Fig. 3.36: Structure of Phosphoglycerate Kinase
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2. 7. Phosphoglycerate Kinase
Catalytic mechanism
The mechanism is nearly the reverse of the first and third reactions in the glycolytic pathway, that of hexokinase and phosphofructokinase. In the first step of the mechanism, an oxyanion of the β phosphate of ADP performs a nucleophilic attack on the carbon 1 phosphoester of 1,3-bisphosphoglycerate. This leaves ATP and 3-phosphoglycerate.
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Fig. 3.37: Catalytic mechanism of Phosphoglycerate Kinase
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2. 7. Phosphoglycerate Kinase
Active site details

The illustration shows the active site of phosphoglycerate kinase. In this case, it is complexed with 3-phosphyglycerate and ATP, the products of the reaction.
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Fig. 3.38: Active site details of Phosphoglycerate Kinase
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2. 8. Phosphoglycerate Mutase
REARRANGEMENT OF 3-PHOSPHOGLYCERATE TO 2-PHOSPHOGLYCERATE

In the eighth reaction of glycolysis, 3-phosphoglycerate is converted to 2-phosphoglycerate by the enzyme phosphoglycerate mutase. This reaction is an isomerization.
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2.8. Phosphoglycerate Mutase
Structure

Each subunit of phosphoglycerate mutase is composed of two subdomains that are made from a central β sheet surrounded by a helices. The active site is located between the two subdomains.
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Fig. 3.39: Structure of Phosphoglycerate Mutase
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2.8. Phosphoglycerate Mutase
Catalytic mechanism

The mechanism of phosphoglycerate mutase involves creation of 2,3-bisphosphoglycerate by a phosphorylated histidine in the enzyme. The histidine-bound phosphate is donated to carbon-2 of 3-phosphoglycerate, then the phosphate of carbon-3 is removed by the histidine, leaving 2-phosphoglycerate and phosphohistidine.
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Fig. 3.40: Catalytic mechanism of Phosphoglycerate Mutase
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2. 8. Phosphoglycerate Mutase
Active site details

2-Phosphoglycerate is complexed with the enzyme, and the active site histidine is highlighted. Note how the histidine is located right between the carbon 2 and carbon 3 positions.
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Fig. 3.41: Active site details of Phosphoglycerate Mutase
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2. 9. Enolase
DEHYDRATION OF 2-PHOSPHOGLYCERATE TO
PHOSPHOENOLPYRUVATE

In the ninth reaction of glycolysis, enolase
catalyzes a dehydration reaction of 2-phosphoglycerate in which water is eliminated, to yield phosphoenolpyruvate.
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2. 9. Enolase
Structure

Enolase is a homodimer with each subunit being a β barrel surrounded by α helices, similar to triose phosphate isomerase. The interface between the two subunits is a flat surface. The active sites are located within a cavity close to the center of each subunit.
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Fig. 3.42: Structure of Enolase
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2. 9. Enolase
Catalytic mechanism

The dehydration is initiated by a base that abstracts a proton from carbon 2 of 2-phosphoglycerate. This forms a carbanion whose electron quickly initiates an enolate formation to carbon 3. The carbon 3 hydroxyl group leaves phosphoenolpyruvate as water that diffuses out of the active site.
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Fig. 3.43:Catalytic mechanism of enolase
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2. 9. Enolase
Active site details

2-phosphoglycerate complexed with enolase. Notice the two bound Mg2+ atoms in each active site (in green). These divalent cations are required for the enzyme to bind the substrate.
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Fig. 3.44: Active site details of Enolase
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2. 10. Pyruvate Kinase
DEPHOSPHORYLATION OF PHOSPHOENOLPYRUVATE TO PYRUVATE

The tenth and final reaction of glycolysis is catalyzed by pyruvate kinase, which converts phosphoenolpyruvate to pyruvate as it transfers the phosphoryl group to ADP to yield ATP.
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2. 10. Pyruvate Kinase
Structure

Pyruvate kinase contains a "cupped hand“ domain made from four separate segments of β sheet surrounded by α helices. The active site is located inside the "cup" of the hand.
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Fig. 3.45: Structure of Pyruvate Kinase
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2.10. Pyruvate Kinase
Catalytic mechanism
The mechanism of pyruvate kinase resembles that of phosphoglycerate kinase. In the first step of the mechanism, an oxyanion of the β phosphate of ADP performs a nucleophilic attack on the carbon 2 phosphoester of phosphoenolpyruvate. This leaves ATP and pyruvate ready to diffuse out of the active site.
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Fig. 3.46: Catalytic mechanism of Pyruvate Kinase
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2. 10. Pyruvate Kinase
Active site details

The active site is situated inside the "cup" of
the hand. In this structure, pyruvate and ADP
are complexed with the enzyme.
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Fig. 3.47: Active site details of Pyruvate Kinase
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Conclusion
The reactions you have just seen are catalyzed by a sequence of 10 enzymes.

Overall, the glycolytic pathway converts one molecule of glucose to two molecules of pyruvate. Two ATP are consumed in the hexokinase and PFK steps. Since two glyceraldehyde-3-phosphate molecules proceed through steps 6–10, the reactions catalyzed by phosphoglycerate kinase and pyruvate kinase generate four ATP, for a net total of two ATP per glucose.
Additional free energy can be extracted from the products of glycolysis: Pyruvate can be further oxidized by the citric acid cycle, and the NADH produced in the glyceraldehyde-3-phosphate dehydrogenase step can be reoxidized to produce ATP by oxidative phosphorylation.
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