Download GOALS FOR LECTURE 7:

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
Carbohydrates
This 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
(6-carbon aldose)
fructose
(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
Glycolysis
All 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 3carbon 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 PO32-.
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 3carbon 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 energygenerating 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 3phosphate 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 2phosphoglycerate. 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. ∆G o ’ for
hydrolysis of an alcohol phosphate (such as 2-phosphoglycerate) is only -3 kcal/mol,
whereas ∆G o ’ 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