GOALS FOR LECTURE 7:

Describe the basic structure of simple and complex carbohydrates

Summarize the early steps in carbohydrate digestion. Describe the consequences and prevalence of adult lactase deficiency.

Distinguish the phases of glycolysis and describe the purpose of each phase.

List the reactions and intermediates of glycolysis. Rationalize the steps and their order based on molecular logic. Identify which pairs of reactions share similar catalytic mechanisms.

List the possible metabolic fates of intermediates generated by glycolysis.

Recommended reading:

TOPIC STRYER, 5th edition DEVLIN, 5th edition

Carbohydrates Ch. 11, 295-306 (not covered)

Glycolysis Ch. 16, 425-443 Ch. 14, 598-610

CarbohydratesThis set of lectures will describe the metabolism of carbohydrates. Metabolism of

fats and proteins will be covered later in the course.

Carbohydrates have the general formula (CH2O)n, carbon + water. Monosaccharides (simple sugars) can have 3 to 8 carbons, and contain multiple hydroxyl groups as well as either an aldehyde group (aldoses) or a ketone group (ketoses).

glucose fructose

(6-carbon aldose) (6-carbon ketose)

7.1

In aqueous solution, one of the hydroxyl groups tends to react with the aldehyde or ketone group, closing the linear molecule into a ring. In an equilibrium mixture, only about 1% of the sugar exists in the open chain form, but the two forms interconvert rapidly. For more information, see PEDS 207, Unit 2.

In the closed ring form, the hydroxyl group on the carbon that carries the aldehyde or ketone can lie either below the plane of the ring or above it. The two positions are called α and β. They also rapidly interconvert, but once one sugar is linked to another, the α or β form is frozen.

The α or β hydroxyl group can react with any hydroxyl group on a second sugar

molecule to form a glycosidic bond, releasing water. Sucrose is a disaccharide formed by an α linkage between carbon 1 of glucose and carbon 2 of fructose. Lactose is formed by a β linkage between carbon 1 of galactose and carbon 4 of glucose. When the hydroxyl group on a carbon that carries a carbonyl is available for such a reaction, the sugar is said to be “reducing”. Sucrose is a nonreducing sugar.

7.2

Oligosaccharides and polysaccharides are formed from multiple sugars linked in a chain by a series of glycosidic bonds. The chemical nature of a polysaccharide is determined by its constituent subunits and the types of linkages between them. For example, a chain of glucose molecules held together by β 1-4 glycosidic bonds is cellulose, a major component of plant cell walls. A chain of glucose subunits held together by α 1-4 glycosidic bonds is α-amylose, or starch.

When carbohydrate-containing food is consumed, polysaccharides and disaccharides are hydrolyzed to their component monosaccharide subunits by digestive enzymes in the saliva in the lumen of the small intestine. The enzymes are stereospecific. α-amylase in the saliva and secreted by the pancreas into the intestine will hydrolyze α 1-4 bonds but not β 1-4 bonds, therefore we can digest starch but not cellulose.

A variety of specific enzymes on the surface of the intestinal cells hydrolyze various disaccharides. Lactose in milk is a major source of nutrition for young mammals including humans, and it is split by β-galactosidase (also called lactase) in the intestine into galactose and glucose. Most adult mammals, including the majority of adult humans, stop secreting this enzyme, and become lactose intolerant. Undigested lactose is osmotically active, drawing water into the large intestine and causing diarrhea. Lactose is readily fermented by the bacteria living in the large intestine, generating CO2 and H2 gas, resulting in bloating and flatulence.

7.3

Two types of glucose carriers enable gut epithelial cells to transfer glucose and other monosaccharides across the gut lining into the bloodstream. Glucose is actively transported into the cell by Na+-driven cotransporters at the apical surface, a process that requires energy (in the form of the Na+ gradient) since glucose is moving from a region where its concentration is low, in the lumen, to a region where its concentration is high, in the intestinal cell cytoplasm. Glucose is released by the cell into the bloodstream by passive transport down its concentration gradient, mediated by a different glucose transporter. Most other cells take up glucose from the bloodstream via passive transport, since their cytoplasmic glucose concentration is lower than the glucose concentration in blood.

ACTIVE TRANSPORT - from the intestinal lumen into gut epithelial cells

PASSIVE TRANSPORT - out of gut epithelial cells, and into other cells

7.4

GlycolysisAll tissues in the body break down glucose to provide energy and intermediates for

other metabolic and biosynthetic pathways. Virtually all sugars can be converted to glucose, so the process of glycolysis is central to carbohydrate metabolism. For cells that lack mitochondria, such as red blood cells, glycolysis is the only available means for generating ATP. Consequently, people with defects in the enzymes that catalyze glycolysis frequently suffer from hemolytic anemia.

During glycolysis, the six-carbon sugar is split into two molecules of pyruvate, which each contain three carbons. Glycolysis proceeds in ten steps. Two molecules of ATP are hydrolyzed to provide energy to drive the early steps, but four molecules of ATP (as well as two molecules of NADH) are produced in the later steps.

There are three phases:

1. Energy investment phase (steps 1-3) uses two molecules of ATP to generate a bisphosphorylated 6-carbon sugar

2. Cleavage phase (steps 4-5) cleaves the 6-carbon sugar into two phosphorylated 3-carbon molecules

3. Energy release phase (steps 6-10) generates 2 molecules of NADH and 4 molecules of ATP

7.5

Step 1: Phosphorylation of glucose.

Glucose is phosphorylated by ATP to form a sugar phosphate. Phosphorylated glucose is not recognized by glucose transporters, so this step effectively traps glucose inside the cell. In most cells, this reaction is catalyzed by the enzyme hexokinase. In liver cells and in the β cells of the pancreas, a different enzyme is used, glucokinase. The different properties of these enzymes are important for the proper regulation of carbohydrate metabolism (which we will discuss below). This is the first energy-consuming step of glycolysis. The rate of entry of glucose into the glycolytic pathway can be regulated at this step.

In these diagrams the symbol represents PO3

2-.

The binding of glucose to hexokinase causes a large conformational change in the enzyme, closing the two halves of the protein together like a clamp.

This conformational change creates a binding site for ATP, and effectively excludes water from the active site. Water at a concentration of 55M would compete very efficiently with glucose for transfer of phosphate. The 5-carbon sugar xylose is also recognized by hexokinase, but because of its smaller size a molecule of water is able to also fit into the active site. In the presence of xylose, hexokinase transfers the terminal phosphate from ATP onto water, releasing ADP, inorganic phosphate, and unmodified xylose.

7.6

Transfer of the phosphate group from ATP to glucose also requires the presence of magnesium.

Step 2: Isomerization of glucose-6-phosphate to fructose-6-phosphate

A readily reversible rearrangement moves the carbonyl oxygen from carbon 1 to carbon 2, transforming the aldose into a ketose.

The enzyme stabilizes the ene-diol intermediate, using concerted general acid-base catalysis. The catalytic residues are lysine (BH+) and glutamate (B’). This is an example of how the special chemical nature of proteins, which can have one acidic group and one basic group closely apposed in the same active site, can generate efficient catalysis.

7.7

Step 3: Phosphorylation of fructose-6-phosphate

The new hydroxyl group on carbon 1 is phosphorylated by ATP. This is the second energy-consuming step, and is the rate-limiting step in glycolysis. Regulation of the enzyme responsible, phosphofructokinase, is the most important control point. The reaction is similar to the hexokinase reaction in step 1, and also requires Mg2+. This reaction makes the substrate almost symmetrical, setting it up to be split in step 4.

Step 4: Cleavage of fructose-1,6-bisphosphate

Aldolase catalyzes the reversible cleavage of the six-carbon sugar into two 3-carbon species, dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This is the only step in glycolysis where a carbon-carbon bond is split. The product G3P can proceed directly through the next steps of glycolysis, but DHAP cannot.

Aldolase forms a covalent bond with its substrate, forming a Schiff base.

7.8

This reaction is the reverse of the aldol condensation frequently used in organic chemistry. Here we see the molecular logic of isomerizing glucose to fructose at step 2: for this cleavage reaction to occur, the carbonyl must be adjacent to the carbon destined to react with the aldehyde.

Step 5: Isomerization of dihydroxyacetone phosphate

DHAP is converted into G3P by triose phosphate isomerase; that is, the ketose is isomerized to the equivalent aldose. This reaction is analogous to step 2, and is also readily reversible.

Triose phosphate isomerase (TIM) stabilizes the ene-diol intermediate, using

concerted general acid-base catalysis. TIM is one of the most efficient enzymes known, approaching “kinetic perfection” (the rate of the reaction is limited by the diffusion rate of the substrate).

7.9

Step 6: Oxidation and phosphorylation of glyceraldehyde-3-phosphate

The two molecules of G3P are oxidized. This is the first part of the energy-generating phase of glycolysis. NAD+ accepts two electrons and one proton to become NADH, and a new high-energy anhydride linkage to phosphate is created.

Glyceraldehyde 3-phosphate dehydrogenase forms a covalent bond to the substrate through a reactive -SH group on the enzyme, and catalyzes its oxidation while still attached. The reactive enzyme-substrate bond is then displaced by an inorganic phosphate ion to produce a high-energy phosphate intermediate, 1,3-BPG.

The human genome encodes at least 46 different copies of glyceraldehyde 3-phosphate dehydrogenase. In comparison, other multicellular organisms such as worms and flies have only 3 or 4. The reason for this is unknown.

7.10

Step 7: Transfer of high-energy phosphate from 1,3-bisphosphoglycerate to ADP

This step generates two molecules of ATP per molecule of glucose, (one molecule of ATP per molecule of 1,3-BPG). At this point, the ATP’s that were “invested” in steps 1 and 3 have been recouped. Combined, step 6 and step 7 oxidize an aldehyde to a carboxylic acid, releasing energy that is stored in the form of NADH and ATP.

Arsenate (AsO43-) closely resembles inorganic phosphate and can substitute for it in

step 6. The product of this reaction, 1-arseno-3-phosphoglycerate, is unstable and decomposes in water to generate 3-phosphoglycerate, without generating an ATP. Thus although the glyceraldehyde-3-phosphate is oxidized, oxidation and phosphorylation reactions are uncoupled, and arsenic is a potent cellular poison.

Step 8: Shift of the phosphate group from carbon 3 to carbon 2

This freely reversible reaction isomerizes 3-phosphoglycerate to 2-phosphoglycerate. The enzyme phosphoglycerate mutase has a phosphoryl group attached to a histidine residue at its active site, which it transfers to form the obligate intermediate 2,3-bisphosphoglycerate. The phosphoryl group is therefore not transferred directly from carbon 3 to carbon 2 within a single substrate molecule; rather, the phosphoryl group of the incoming molecule of 3-phosphoglycerate ends up on carbon 2 of the next substrate whose conversion is catalyzed by the enzyme.

7.11

Occasionally, 2,3-BPG dissociates from the enzyme, leaving it in an inactive form. For this reason, the cell needs to maintain trace levels of 2,3-BPG to regenerate the phosphoenzyme by the reverse reaction. 2,3-BPG is synthesized for this purpose by a separate pathway, involving isomerization of 1,3-BPG by bisphosphoglycerate mutase. This reaction is particularly important in red blood cells, where 2,3-BPG has a role in regulating the affinity of hemoglobin for oxygen.

Step 9: Dehydration of 2-phosphoglycerate

The removal of water from 2-phosphoglycerate changes the relatively low-energy phosphate ester linkage to a high-energy enol phosphate linkage. This reaction is readily reversible.

7.12

This critical step puts the phosphate group at a very high energy state. ∆Go’ for

hydrolysis of an alcohol phosphate (such as 2-phosphoglycerate) is only -3 kcal/mol, whereas ∆Go’ for the hydrolysis of an enol phosphate (such as phosphoenolpyruvate) is -14.8 kcal/mol. This large difference is due to the difference in the oxidation states of the eventual products: a ketone is more oxidized than an alcohol, and therefore is considered to be at lower energy. This sets up a big energy payoff at step 10....

Step 10: Formation of pyruvate

In the last step of glycolysis, the high-energy enol phosphate is transferred to ADP, generating another molecule of ATP (two molecules of ATP per original molecule of glucose). This step is effectively irreversible, and pyruvate kinase is the third regulated enzyme of glycolysis.

7.13

Fates of pyruvate

In most animal and plant cells, glycolysis is a prelude to the final stage of energy production, which occurs in the mitochondria. Pyruvate is imported into mitochondria, where it is converted into acetyl-CoA. Acetyl-CoA is an important building block in biosynthetic reactions. It is also a major fuel for the TCA cycle. Within the mitochodria, acetyl-CoA is completely oxidized by the TCA cycle and oxidative phosphorylation into CO2 and water, generating large amounts of ATP (30 mol/mol glucose, compared to 2 mol/mol for glycolysis). Reducing equivalents present in the cytoplasm as NADH (generated in step 6) can be shuttled into the mitochondria by two different mechanisms that regenerate NAD+, and the resulting reducing equivalents in the mitochondria can be used directly in oxidative phosphorylation to generate ATP. These pathways require molecular oxygen (O2), and will be detailed in the next set of lectures on aerobic metabolism.

In human cells that lack mitochondria (such as red blood cells and the lens cells in the eye), or when there is little or no molecular oxygen available (such as in skeletal muscle during vigorous exercise), pyruvate remains in the cytoplasm. Under these conditions, glycolysis can be limited by the amount of NAD+ available for the oxidation of glyceraldehyde-3-phosphate in step 6. NADH generated in this step can be to reduce pyruvate to lactate, a reaction catalyzed by lactate dehydrogenase, regenerating NAD+.

Lactic acid can accumulate in muscle, causing cramps. Most lactate eventually

diffuses into the bloodstream. In the liver, lactate can be reconverted to glucose by the process of gluconeogenesis, which will be described later. Lactate is reoxidized to pyruvate in heart muscle, and used to make ATP in the TCA cycle. As blood pH drops, excess protons bind to hemoglobin, reducing its affinity for O2 (the Bohr effect).

Excessive lactate production can result in lactic acidosis, where blood pH drops significantly. High blood lactate can be a sign of general tissue hypoxia, such as during shock or pulmonary failure.

7.14

In yeast and some bacteria, pyruvate is converted first into acetaldehye by pyruvate decarboxylase, and then reduced to ethanol by NADH. Like the production of lactate, the conversion of glucose to ethanol involves no net oxidation and can occur in the absence of O2.

Finally, pyruvate can be used in biosynthetic pathways; e.g. to make glucose via gluconeogenesis, or to make alanine or aspartate.

Alternate fates of glycolytic intermediates

Glycolytic intermediates are used in the synthesis of many other cellular constituents, including amino acids, lipids, and nucleotides.

7.15

Summary of glycolysis

7.16