1. Basic Concepts of Cellular Metabolism and Bioenergetics
Intermediary Metabolism
The Chemistry of Metabolism
Concepts of Bioenergetics
Experimental Study of Metabolism

2. Metabolism Metabolism
The summation of all chemical reactions in an organism.
Metabolic differences are best studied by dividing all life into two categories.
Autotrophs - organisms that use atmospheric CO2 as their sole source of carbon.
Heterotrophs - life forms that obtain energy by ingesting complex carbon compounds .

3. Intermediary metabolism
Metabolism relies on thousands of sequential enzymatically controlled reactions.
Intermediary metabolism. Products from one reaction often become the reactant for the next - metabolites.
Pathway. A series of reactions with a specific purpose. Linear - Glycolysis
Branched - Amino acid biosynthesis
Cyclic - Citric Acid Cycle
Spiral - Fatty acid degradation

4. Intermediary metabolism
Two paths of metabolism:
Catabolism
Degradation path. Complex organic molecules are degraded to simpler species. Production of energy.
Anabolism
Construction path. Biosynthesis of more complex organic compounds. Requires energy.

5. Energy, ATP and the movement of phosphate
phosphoenolpyruvate P
ADP
1,3-bisphosphoglycerate P
creatine phosphate P
Energy
ATP
glucose-1-phosphate P
fructose-6-phosphate P
ADP
glucose-6-phosphate P

6. ATP ATP adenosine triphosphate
a nucleotide composed of three basic units.
adenine
phosphate chain
ribose CH 2 O OH N NH P -

7. ATP and ADP It takes energy to put on the third phosphate. Energy is
released when
it is removed.
ADP - ATP
conversions act
as a major
method of
transferring
energy. CH 2 O OH N NH P O- -
ADP CH 2 O OH N NH P -
ATP

8. Catabolic stages of metabolism
Stage I
Breakdown of macromolecules into their building blocks. No useful energy.
Stage II
Oxidation of Stage I products to acetyl CoA. Limited energy production.
Stage III
Oxidation of acetyl CoA to CO2 and H2O and energy.

9. Overview of catabolic processes
Carbohydrates
Fats
Proteins
Simple Sugars
Fatty acids
Amino acids
Stage 1
Stage 2
Stage 3
Glycolysis
ATP
Pyruvate
Acetyl CoA
Citric acid cycle
Oxidative phosphorylation
ATP

10. Overview of catabolic metabolism
ATP
ADP + Pi
protein
amino acids
polysaccharides
hexoses
pentoses
ADP + Pi
ATP
ADP + Pi
ATP
lipids
fatty acids
ADP + Pi
ADP + Pi
ATP
ADP + Pi
ADP + Pi
ATP
ATP
pyruvate
ATP
urea
urea
cycle
ADP + Pi
acetyl-CoA O2
ATP
electron transport
chain
oxidative
phosphorylation e-
citric acid
cycle
CO2
ATP

11. Stage one Hydrolysis of food into smaller subunits. Handled by
the digestive
system.

12. Stage one Salivary glands Secrete amylase - digests starch. Stomach
Secretes HCl - denatures protein and pepsin.
Pancreas
Secretes proteolytic enzymes and lipases - degrades proteins and fats.

13. Stage one Liver and gallbladder Deliver bile salts.
- emulsify fat globules - easier to digest.
Small intestine
Further degradation.
Produces amino acids, hexose sugars, fatty acids and glycerol.
Moves materials into blood for transport to cells.

14. The chemistry of metabolism
Six categories of biochemical reactions have been identified.
Oxidation-reduction
Group-transfer
Hydrolysis
Nonhydrolytic cleavage
Isomerization and rearrangement
Bond formation reactions using energy from ATP

15. Oxidation-Reduction Most common of all metabolic reactions.
There are always two reactant molecules.
They are readily identified by the transfer of hydrogen atoms.
Enzymes involved in these reactions are oxidoreductases (dehydrogenases).
AH2 + B A + BH2

16. Oxidation-Reduction
When an atom or group is oxidized, some other species must accept the electrons.
Many reactions are coupled to the coenzyme pairs.
NAD+ / NADH
NADP+ / NADPH
FAD / FADH2

17. Coenzymes used in metabolism
NAD NADH
Oxidized form Reduced form
of nicotinamide adenine dinucleotide.
Used in REDOX reactions.
It is a derivative of ADP and the vitamin nicotinamide.
The reactive site is located on the nicotinamide portion of NAD+.

18. Coenzymes used in metabolism
reactive
site
nicotinamide
adenine
ribose

19. NAD+

20. Coenzymes used in metabolism
Example reactions of NAD+
General reaction
Specific example - ethanol
CH3CH2OH + NAD+ H
CH3C=O + NADH + H+ OH O R C H
+ NAD + R C H
+ NADH + H + H
alcohol
dehydrogenase

21. Coenzymes used in metabolism
FAD - flavin adenine dinucleotide.
Another major electron carrier used in metabolism.
It involves a two electron transfer so it picks up two hydrogen.
FAD FADH2

22. Coenzymes used in metabolism
FAD
ribose
adenine
riboflavin
Reactive site
is highlighted

23. FAD

24. Coenzymes used in metabolism
FAD typically reacts with different substrates than NAD+.
FAD is often involved in oxidation reactions in which a -CH2 - CH2 - portion is oxidized to a double bond.
O O
|| ||
CH3CH2CH2-C-S-CoA CH3CH=CHC-S-CoA
FAD FADH2

25. Group-Transfer
Reactions that involve moving a chemical functional group.
Intermolecular. Transfer from one molecule to another.
Intramolecular. Movement from one location to another on the same molecule.
Phosphate is one of the most important groups that is transferred.

26. Group-Transfer Another common group to transfer is acyl group. O
Coenzyme A (CoASH) will form a thioester linkage to this group, making it more active.
R - C O

27. Acetyl - coenzyme A This molecule serves as the carrier
pantothenate
unit
phosphorylated
ADP NH 2 O O H
CH3 O O N N
C-CH2-CH2-N-C-C-C-CH2 O P O P O - N H HO
CH3 O O - CH N
H-N 2 O
CH2-CH2
Sulfhydyl
group S OH O
CH3C P - O O O O -
acetate
This molecule serves as the carrier
for the small molecules from digestion.

28. Acetyl - coenzyme A

29. Hydrolysis
Water is used to split a single molecule into two separate molecules.
Most common types of bonds to split
Esters - fats
Amides - proteins
Glycosidic - carbohydrates

30. Hydrolysis
Carbohydrates O OH H CH 2 HO
+ H2O
enzyme

31. Hydrolysis Proteins + H | H2NCCOOH R R’ H O | || H2N - C - C -
| ||
H2N - C - C -
N - C - COOH
| |
H R’
enzyme
+ water

32. Hydrolysis Fats + 3 H2O C R O HO O C R H R’ R’’ OH C H C R’ O HO + C

33. Nonhydrolytic cleavage
A class of reactions where molecules are split without the use of water.
Lyases - Enzymes that accomplish this task.
fructose-1, dihydroxyacetone glyceraldehyde
bisphosphate phosphate phosphate

34. Isomerization and rearrangement
This category involves two kinds of chemical transformations:
Intermolecular hydrogen atom shifts to the location of a double bond. Most prominent example is the aldose-ketose isomerization.
Intramolecular rearrangements of functional groups. These are rare.

35. Isomerization and rearrangementC CH 2 OH H HO O
CH2OH
aldose
cis-enediol
intermediate
ketose

36. Bond formation reactions using energy
Category of biochemical bond formation reactions. All require an energy source.
spontaneous
DHase
isocitrate oxalosuccinate -ketoglutarate

37. Concepts of bioenergetics
Standard free energy change - Go
The energy change occurring when a reaction, under standard conditions, proceeds from start to equilibrium.
Equilibrium
A + B C + D
K’eq =
[ C ] [ D ]
[ A ] [ B ]

38. Standard free energy changes
Go can be related to the equilibrium expression by:
Go’ = RT log K’eq
where
Go’ standard free energy change
R gas constant, J/mol
T temperature, kelvin
K’eq equilibrium constant

39. Standard free energy changes
These types of measurements can be made by mixing the reactants at 1 molar, 25oC and a pH of 7 in a test tube.
Unfortunately, they do not agree well with the conditions of a living cell.
They do provide an estimate for comparing energy requirements among the many reactions in a cell.

40. Standard free energy changes
Go’ = 0 System at equilibrium, no release or requirement of energy.
Go’ < 0 Reaction releases energy as it approaches equilibrium.
Go’ > 0 Reaction requires that energy be added to proceed in the direction indicated.

41. Experimental measurement of Go’
As an example, let’s determine Go’ for the isomerization of glucose-6-phosphate to fructose-6-phosphate.
To start, solutions are mixed that result in an initial concentration of one molar for each species at standard conditions.
At equilibrium we have:
[ glucose-6-phosphate ] = 1.33 M
[ fructose-6-phosphate ] = 0.67 M

42. Experimental measurement of Go’
K’eq = 0.67 M / 1.33 M
= 0.50
Go’ = (-2.303)(8.315 J/mol)(298 K) log(0.5)
= J/mol = +1.7 kJ/mol
This indicates that energy is required for glucose-6-phosphate to be converted to fructose-6-phosphate -- it is not spontaneous.

43. Energy from ATP We can conduct a similar experiment using ATP and ADP:
ATP + H2O ADP + Pi
After mixing and allowing to reach equilibrium, we find that the concentration of ATP is too low to measure.
We can’t directly obtain Go’ but at least we know that it must be negative.

44. Energy from ATP
Using a coupled reaction, it is possible to measure the Go’ for ATP.
Go’ kJ/mol
glucose + ATP glucose-6-phosphate + ADP -16.7
glucose-6-phosphate + H2O glucose + Pi -13.8
Sum: ATP + H2O ADP + Pi
This is a relatively large amount of useful chemical energy.

45. Experimental study of metabolism
To understand a pathway, one must know all of the details of each step.
Characterization of each enzyme and coenzyme.
Identification of the chemical pathway, including the substrate, intermediates, products and types of reaction.
Identification of molecules and conditions that regulate the overall rate of the pathway.

46. Experimental study of metabolism
Whole organisms.
One can introduce radiolabeled materials and measure any labeled waste products.
Tissue slices and cells.
These have been used to uncover metabolic details. The citric acid cycle was characterized using this approach.
Cell-free extracts.
Cells are homogenized in a buffer to release cell components for study.