Bioenergetics and Metabolism

1
Glycolysis 1Glycolysis consists of two stages,
an ATP investment stage, and an ATP earnings
stage
Bioc 460 Spring 2008 - Lecture 25 (Miesfeld)
Metabolism of glucose by yeast under anaerobic
conditions leads to the production of ethanol and
CO2
Lactate build-up can limit exercise
2
Key Concepts in Glycolysis

• Metabolic pathways consist of a series of
  enzymatic reactions that share reactants and
  products. Metabolic flux (rate of metabolite
  interconversion) is highly-regulated by both
  enzyme activity and substrate availability.
  Nutrient availability affects metabolic flux.
• Glycolysis is an ancient pathway that cleaves
  glucose (C6H12O6) into two molecules of pyruvate
  (C3H3O3). Under aerobic conditions, the pyruvate
  is completely oxidized by the citrate cycle to
  generate CO2, whereas, under anaerobic (lacking
  O2) conditions, it is either converted to
  lactate, or to ethanol CO2 (fermentation).
• The glycolytic pathway consists of ten enzymatic
  steps organized into two stages. In Stage 1, two
  ATP are invested to prime the pump, and in
  Stage 2, four ATP are produced to give a net ATP
  yield of 2 ATP/glucose.
• Three glycolytic enzymes catalyze highly
  exergonic reactions (?Gltlt0) which drive metabolic
  flux through the pathway these enzymes are
  regulated by the energy charge in the cell (ATP
  requirements). The three enzymes are hexokinase,
  phosphofructokinase 1, and pyruvate kinase.

3
Glucose metabolism in the liver before breakfast
Glucagon signaling in liver cells activates both
a catabolic pathway (glycogen degradation) and an
anabolic pathway (gluconeogenesis), while at the
same time inhibiting the catabolism of glucose by
the glycolytic pathway.
4
Glucose metabolism in the liver after breakfast
Within an hour of eating a bowl of cereal and
drinking a cup of fruit juice, your insulin
levels increase due to elevated blood glucose
causing activation of the insulin signaling
pathway and stimulation of glucose uptake,
glycogen synthesis, and an increase in glucose
catabolism by the glycolytic pathway.
5
Metabolic pathways are linked sets of enzymatic
reactions that share common intermediates
Remember that substrate concentration and enzyme
activity levels affect flux through metabolic
pathways
6
There are six major groups of metabolic pathways
in nature

• Anaerobic and aerobic respiration
• Photosynthesis and carbon fixation
• Carbohydrate metabolism
• Lipid metabolism
• Amino acid metabolism
• Nucleotide metabolism

• Metabolic pathways are highly interdependent and
  exquisitely controlled by substrate availability
  and enzyme activity levels.
• Key to understanding metabolic integration in
  terms of nutrition, exercise, and disease (e.g.,
  diabetes and obesity) is learning how metabolic
  flux between pathways is regulated and
  controlled.

7
http//www.expasy.org/cgi-bin/show_thumbnails.pl
Should you memorize this chart?
8
Hierarchical Nature of Metabolism Four classes
of macromolecules (proteins, nucleic acids,
carbohydrates, and lipids) Six primary
metabolite groups (amino acids, nucleotides,
fatty acids, glucose, pyruvate, acetyl CoA)
Seven small biomolecules (NH4, CO2, NADH,
FADH2, O2, ATP, H2O)
Note that you can download a PDF file of this
metabolic map from the study guides page on the
website.
9
The six major groups of pathways can be
subdivided into those responsible for energy
conversion, and those involved in the synthesis
and degradation of macromolecules. Moreover,
the major groups themselves consist of several
metabolic modules, all of which will be examined
individually.
10
We will use the divide and conquer strategy to
study metabolism, starting with energy conversion
pathways, and then examining the synthesis and
degradation of carbohydrates, lipids, and amino
acids. Nucleotide metabolism is taught in other
courses. The last two lectures in this course
(lectures 40 and 41) cover metabolic regulation
using diet, exercise, and diabetes as conceptual
themes.
11
Four central questions to keep in mind when you
study each metabolic pathway

• 1. What does the pathway accomplish for the cell?
• 2. What is the overall net reaction of the
  pathway?
• 3. What are the key regulated enzymes in the
  pathway?
• 4. What are examples of this pathway in real
  life?

12
Glycolysis Lets Answer the Four Questions

• What does glycolysis accomplish for the cell?
• Generates a small amount of ATP which is critical
  under anaerobic conditions.
• Generates pyruvate, a precursor to acetyl CoA,
  lactate, and ethanol (in yeast).
• What is the overall net reaction of glycolysis?
• Glucose 2NAD 2ADP 2 Pi ?
• 2 pyruvate 2NADH 2H 2ATP 2H2O
• ?Gº -35.5 kJ/mol

13
The Four Metabolic Pathway Questions

• 3. What are the key regulated enzymes in
  glycolysis?
• Hexokinase, Phosphofructokinase 1, Pyruvate
  kinase
• 4. What are examples of glycolysis in real
  life?
• Glycolysis is the sole source of ATP under
  anaerobic conditions which can occur in animal
  muscle tissue during intense exercise.
  Fermentation also relies on glycolysis which is a
  process that is used to make alcoholic beverages
  when yeast cells are provided glucose without
  oxygen.

14
Where does glycolysis fit into the metabolic map?
Glycolysis is a central pathway that takes
glucose generated by carbohydrate metabolism and
converts it to pyruvate. Under aerobic
conditions, the pyruvate is oxidized in the
citrate cycle which generates reducing power for
redox reactions in the electron transport system
that result in ATP production by oxidative
phosphorylation.
15
Glycolysis takes place entirely in the cytosol,
whereas, pyruvate oxidation occurs in the
mitochondrial matrix where ATP is generated.
Oxygen is not required for glycolysis in the
cytosol (anaerobic) but it is necessary for
aerobic respiration in the mitochondrial matrix
where the O2 serves as the terminal electron
acceptor.
16
The complete oxidation of glucose to CO2 and H2O
is highly favorable and releases a large amount
of energy that can be harnessed for ATP synthesis

• Glucose (C6H12O6) 6O2 ? 6CO2 6H2O
• ?Gº -2,840 kJ/mol?G -2,937 kJ/mol
• ?Gº for ATP synthesis -30.5 kJ/mol
• ?G for ATP synthesis -50 kJ/mol
• Theoretical maximum yield 60 ATP/glucose
• Actual yield 32 ATP/glucoseWhy are only 32
  ATP generated out of a possible 60 ATP?

17
Pyruvate can also be converted anaerobically to
ethanol and CO2 by fermentation in some
micoroorganisms, or converted to lactate
18
For every mole of glucose entering glycolysis,
two moles of glyceraldehyde-3-P (GAP) are
metabolized to pyruvate, generating in the
process a net 2 ATP and 2 NADH.The NADH is a
source of reducing power for the cell.
Overview of the Glycolytic Pathway
19
The two stages of glycolysis
The ATP investment stage generates the high
energy intermediate glyceraldehyde-3-P (GAP)
which is then oxidized to produce NADH and
1,3-bisphosphoglycerate. The next four reactions
lead to the production of FOUR total ATP because
each glucose molecule results in the production
of TWO pyruvate. The net yield of ATP in
glycolysis is therefore TWO ATP.
20
Stage 1
Investment of 2 ATP Production of 2
Glyceraldehyde-3-P (GAP) The two highly
regulated steps are hexokinase and
phosphofructokinase 1 (both respond directly or
indirectly to energy charge).
21
Each molecule of GAP
Stage 2
Reducing power is captured in the form of NADH
this is a critical step. Phosphoglycerate
kinase and pyruvate kinase catalyze a substrate
level phosphorylation reaction yielding 4 ATP (2
net ATP). The two pyruvate molecules are
further metabolized.
22
No loss of carbons or oxygen in glycolysis
The six carbons and six oxygens present in
glucose are stoichiometrically conserved by
glycolysis in the two molecules of pyruvate that
are produced. Hydrogen atoms in glucose are lost
as H2O molecules and in the reduction of NAD.
23
Chemical features of the glycolytic reactions

• Ten enzymatic reactions
• primarily bond rearrangements
• phosphoryl transfer reactions
• isomerizations
• an aldol cleavage
• an oxidation
• a dehydration
• Ideally, you should know the names of all ten
  enzymes and the reactants and products. The
  names describe the metabolite structures, draw
  them if you like, or visualize them in your head.
• At the very minimum, you need to know which steps
  the ATP hydrolysis and synthesis takes place, the
  net reaction of glycolysis, and the three key
  enzymes the control glycolytic flux.

24
Free energy changes for the ten glycolytic
reactions
?Gº -35.5 kJ/mol
?G -72.4 kJ/mol
25
Reaction 1 Phosphorylation of glucose by
hexokinase or glucokinase
Hexokinase is found in all cells. A related
enzyme with same enzymatic activity, glucokinase,
is present primarily in liver and pancreatic
cells.
26
Hexokinase binds glucose through an induced fit
mechanism that excludes H2O from the enzyme
active site and brings the phosphoryl group of
ATP into close proximity with the C-6 carbon of
glucose
27
Hexokinase is feedback inhibited by glucose-6-P
which binds to a regulatory site in the amino
terminus of the enzyme
Why does it make sense that hexokinase is
feedback inhibited by glucose-6-P when energy
charge in the cell is high?
28
Reaction 2 Isomerization of glucose-6-P to
fructose-6-P by phosphoglucose isomerase
Phosphoglucose isomerase (phosphohexose
isomerase) interconverts an aldose (glucose-6-P)
and a ketose (fructose-6-P) through a complex
reaction mechanism that involves opening and
closing of the ring structure.
29
Reaction 3 Phosphorylation of fructose-6-P to
fructose-1,6-BP by phosphofructokinase 1
Reaction 3 is the second ATP investment reaction
in glycolysis and involves the coupling of an ATP
phosphoryl transfer reaction catalyzed by the
enzyme phosphofructokinase 1 (PFK-1). This is a
key regulated step in the glycolytic pathway
because the activity of PFK-1 is controlled by
numerous allosteric effectors (positive and
negative).
30
Reaction 4 Cleavage of fructose-1,6-BP by
aldolase to generate glyceraldehyde-3-P and
dihydroxyacetone-P
The splitting of fructose-1,6-BP into the triose
phosphates glyceraldehyde-3-P and
dihydroxyacetone-P is the reaction that puts the
lysis in glycolysis (lysis means splitting).
31
Reaction 5 Isomerization of dihydroxyacetone-P
to glyceraldehyde-3-P by triose phosphate
isomerase
The original TIM barrel structure
Glyceraldehyde-3-P, rather than
dihydroxyacetone-P, is the substrate for reaction
6 in the glycolytic pathway, making this
isomerization necessary.
32
STAGE 2 ATP EARNINGS

• Three key features of the very important stage 2
  reactions
• Two substrate level phosphorylation reactions
  catalyzed by the enzymes phosphoglycerate kinase
  and pyruvate kinase generate a total of 4
  ATP/glucose (net yield of 2ATP) in stage 2 of
  glycolysis.
• An oxidation reaction catalyzed by
  glyceraldehyde-3-P dehydrogenase generates 2 NADH
  molecules that can be shuttled into the
  mitochondria to produce more ATP by oxidative
  phosphorylation.
• Reaction 10 is an irreversible reaction that must
  be bypassed in gluconeogenesis by two separate
  enzymatic reactions catalyzed by pyruvate
  carboxylase and phosphoenolpyruvate carboxykinase

33
Reaction 6 Oxidation and phosphorylation of
glyceraldehyde-3-P by glyceraldehyde-3-P
dehydrogenase to form 1,3-bisphosphoglycerate
The glyceraldehyde-3-P dehydrogenase reaction is
a critical step in glycolysis because it uses the
energy released from oxidation of
glyceradehyde-3-P to drive a phosphoryl group
transfer reaction using inorganic phosphate (Pi)
to produce 1,3-bisphosphoglycerate.
34
Reaction 7 Generation of ATP by phosphoglycerate
kinase in the conversion of1,3-bisphosphoglycerat
e to 3-phosphoglycerate
Phosphoglycerate kinase catalyzes the payback
reaction in glycolysis because it replaces the 2
ATP that were used in stage 1 to prime the
glycolytic pathway. Remember, this occurs twice
for every glucose that entered glycolysis. This
is an example of a substrate level ADP
phosphorylation reaction, i.e., ATP synthesis
that is not the result of aerobic respiration or
photophosphorylation.
35
Reaction 8 Phosphoryl shift by phosphoglycerate
mutase to convert 3-phosphyglycerate to
2-phosphoglycerate
The purpose of reaction 8 is to generate a
compound, 2-phosphoglycerate, that can be
converted to phosphoenolpyruvate in the next
reaction, in preparation for a second substrate
level phosphorylation to generate ATP.
36
The mechanism of this highly reversible reaction
requires a phosphoryl transfer from a
phosphorylated histidine residue (His-P) located
in the enzyme active site
The metabolic intermediate 2,3-BPG can diffuse
out of active site before it is converted to
2-phosphoglycerate. Remember that 2,3-BPG is
important in the regulation of oxygen binding by
hemoglobin.
37
Reaction 9 Dehydration of 2-phosphoglycerate by
enolase to form phosphoenolpyruvate (PEP)
The standard free energy for this reaction is
relatively small (?Gº 1.7 kJ/mol) but it
traps the phosphate group in an unstable enol
form, resulting in a dramatic increase in the
phosphoryl transfer potential of the triose
sugar. Standard free energy change for phosphate
hydrolysis in 2-phosphoglycerate is ?Gº -16
kJ/mol, whereas the standard freen energy change
for phosphate hydrolysis of phosphoenolpyruvate
it is an incredible ?Gº -62 kJ/mol !
38
Reaction 10 Generation of ATP by pyruvate kinase
when phosphoenolpyruvate is converted to pyruvate
The second of two substrate level phosphorylation
reactions in glycolysis that couples energy
released from phosphate hydrolysis (?Gº -62
kJ/mol) to that of ATP synthesis (?Gº 30.5
kJ/mol). Unlike phosphoenolpyruvate, pyruvate is
a stable compound in cells that is utilized by
many other metabolic pathways.
39
Fill in the names of all glycolytic enzymes and
metabolites based only on the chemical structures