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

1

Basic Concepts of Cellular Metabolism and Bioenergetics

Basic Concepts of Cellular Metabolism and Bioenergetics

Intermediary Metabolism

The Chemistry of Metabolism

Concepts of Bioenergetics

Experimental Study of Metabolism

Intermediary Metabolism

The Chemistry of Metabolism

Concepts of Bioenergetics

Experimental Study of Metabolism

2

MetabolismMetabolism

MetabolismMetabolismThe summation of all chemical reactions in an organism.

Metabolic differences are best studied by dividing all life into two categories.

AutotrophsAutotrophs - organisms that use atmospheric CO2 as their sole source of carbon.

HeterotrophsHeterotrophs - life forms that obtain energy by ingesting complex carbon compounds .

3

Intermediary metabolismIntermediary metabolism

Metabolism relies on thousands of sequential enzymatically controlled reactions.

Intermediary metabolism.Intermediary metabolism. Products from one reaction often become the reactant for the next - metabolites.

Pathway.Pathway. A series of reactions with a specific purpose. Linear - GlycolysisLinear - Glycolysis

Branched - Amino acid Branched - Amino acid biosynthesisbiosynthesis

Cyclic - Citric Acid CycleCyclic - Citric Acid Cycle

Spiral - Fatty acid degradationSpiral - Fatty acid degradation

4

Intermediary metabolismIntermediary metabolism

Two paths of metabolism:

CatabolismCatabolismDegradation path. Complex organic molecules are degraded to simpler species. Production of energy.

AnabolismAnabolismConstruction path. Biosynthesis of more complex organic compounds. Requires energy.

5

Energy, ATP and themovement of phosphate

Energy, ATP and themovement of phosphate

ATP

ADP

ADP

phosphoenolpyruvatephosphoenolpyruvate

1,3-bisphosphoglycerate1,3-bisphosphoglycerate

creatine phosphatecreatine phosphate

glucose-1-phosphateglucose-1-phosphate

fructose-6-phosphatefructose-6-phosphate

glucose-6-phosphateglucose-6-phosphate

P

En

erg

y

P

P

P

P

P

6

ATPATP

ATPATP adenosine triphosphateadenosine triphosphatea nucleotide composed of three basic units.

adenine

phosphate chain

ribose

CH2 O

OH OH

N

NN

N

NH2

OPOPOPO-

O

O-

O

O-

O

O-

7

ATP and ADPATP and ADP

CH 2 O

OH OH

N

NN

N

NH 2

OPOPO-

O

O-

O

O-

ADP

CH 2 O

OH OH

N

NN

N

NH 2

OPOPOPO-O

O-

O

O-

O

O-

ATP

It takes energyto put on thethird phosphate.

Energy isreleased whenit is removed.

ADP - ATP conversions actas a major method oftransferringenergy.

8

Catabolic stages of metabolismCatabolic stages of metabolism

Stage IStage IBreakdown of macromolecules into their building blocks. No useful energy.

Stage IIStage IIOxidation of Stage I products to acetyl CoA. Limited energy production.

Stage IIIStage IIIOxidation of acetyl CoA to CO2 and H2O and energy.

9

Overview of catabolic processes

Overview of catabolic processes

CarbohydratesCarbohydrates FatsFatsProteinsProteins

Simple SugarsSimple Sugars Fatty acidsFatty acidsAmino acidsAmino acids

PyruvatePyruvate

Acetyl CoAAcetyl CoA

Oxidative phosphorylationOxidative phosphorylation

ATP

ATP

Citric acid cycleCitric acid cycle

Stage 1

Stage 2

Stage 3

GlycolysisGlycolysis

10

Overview of catabolic metabolismOverview of catabolic metabolism

acetyl-CoA

pyruvate ATP

ADP + Pi

polysaccharides

hexosespentoses

ADP + Pi

ATP

ADP + Pi

ATP

ADP + Pi

ATPATP

ADP + Pi

ADP + Pi

ATP

lipids

fatty acids

ATP

ADP + Pi

protein

amino acids

citric acidcycle

ureacycle ATP

ADP + Pi

urea

CO2

electron transportchain

oxidative phosphorylation

O2

ATP

e-

11

Stage oneStage one

Hydrolysis of food into smaller subunits.

Handled bythe digestivesystem.

Handled bythe digestivesystem.

12

Stage oneStage one

Salivary glandsSalivary glandsSecrete amylase - digests starch.

StomachStomachSecretes HCl - denatures protein and pepsin.

PancreasPancreasSecretes proteolytic enzymes and lipases - degrades proteins and fats.

13

Stage oneStage one

Liver and gallbladderLiver and gallbladderDeliver bile salts.- emulsify fat globules - easier to digest.

Small intestineSmall intestineFurther degradation.Produces amino acids, hexose sugars, fatty acids and glycerol.Moves materials into blood for transport to cells.

14

The chemistry of metabolismThe 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-ReductionOxidation-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-ReductionOxidation-Reduction

When an atom or group is oxidized, some other species must accept the electrons.

Many reactions are coupled to the coenzyme pairs.

NADNAD++ / NADH / NADH

NADPNADP++ / NADPH / NADPH

FAD / FADHFAD / FADH22

17

Coenzymes usedin metabolism


NADNAD++ NADH NADHOxidized 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



OCH2

OH OH

OPO

O-

O

OCH2

OH OH

OPO

O-N

NN

N

NH2

N+

C

O

NH2

reactivesite

nicotinamide

adenine

ribose

19

NAD+NAD+

20



Example reactions of NAD+

General reactionGeneral reaction

Specific example - ethanolSpecific example - ethanol

CH3CH2OH + NAD+

H CH3C=O + NADH + H+

R COHH

HHH + NAD+ R C

OH + NADHH + HH++

alcoholdehydrogenase

21



FAD - flavin adenine dinucleotide.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



FAD

OCH2

OH OH

OPO

O-N

NN

N

NH2

O

C HH

CH OH

CH OH

CH OH

CH H

N

NH3C

H3C N

NH

O

O

ribose

adenine

riboflavin

Reactive siteis highlighted

23

FADFAD

24



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-CoAFAD FADH2

25

Group-TransferGroup-Transfer

Reactions that involve moving a chemical functional group.

Intermolecular.Intermolecular. Transfer from one molecule to another.

Intramolecular.Intramolecular. Movement from one location to another on the same molecule.

Phosphate is one of the most important groups that is transferred.

26

Another common group to transfer is acyl group.

Coenzyme A (CoASH) will form a thioester linkage to this group, making it more active.

Group-TransferGroup-Transfer

R - C

O

27

Acetyl - coenzyme AAcetyl - coenzyme A

This molecule serves as the carrier

for the small molecules from digestion.

phosphorylated ADP

pantothenateunit

acetate

CH2 O

O OH

N

NN

N

NH2

OPOPO

O-

O

O-

P O-O

O-

C-CH2-CH2-N-C-C-C-CH2

OO H

CH3HO

CH3

HH-N

CH2-CH2

S

CH3C O

Sulfhydylgroup

O

28

Acetyl - coenzyme AAcetyl - coenzyme A

29

HydrolysisHydrolysis

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


CarbohydratesCarbohydrates

OOH

H

H

H

H

OH

CH 2 OH

H

OH

OH OH

HH

H

OH

CH 2 OH

H

OH

O

OOH

H

H

H

H

OH

CH 2 OH

H

OH

OH OH

HH

H

OH

CH 2 OH

H

OHOH HO

+ H2O

enzyme

31


ProteinsProteins

H |

H2NCCOOH

| R

+

H |

H2NCCOOH

| R’

H O | ||

H2N - C - C -

| R

H |N - C - COOH | |H R’ enzyme

+ water

32


FatsFats

O C R

O

C

H

H

O C R’

O

CH

O C R’’

O

C

H

H

OHC

H

H

OHCH

OHC

H

H

C R’’

O

HO

++ 3 H2OC R’

O

HO

C R

O

HO

33

Nonhydrolytic cleavageNonhydrolytic cleavage

A class of reactions where molecules are split without the use of water.

LyasesLyases - Enzymes that accomplish this task.

CH2OPO32-

C O

C

C

C

CH2OPO32-

H

H

H

HO

HO

HO

CH2OPO32-

C O

CH2OH

C

C

CH2OPO32-

OH

HO H+aldolase

fructose-1,6- dihydroxyacetone glyceraldehydebisphosphate phosphate 3-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



C

C

C

C

C

CH 2 OH

OH

OH

H

OHH

HO

H

H

OH

C

C

C

C

C

CH 2 OH

OH

OH

H

OH

HO

H

H

C

C

C

C

CH2OH

CH 2 OH

OH

OH

H

O

HO

H

H

OHH

aldosecis-enediol

intermediate ketose

36

Bond formation reactionsusing energy

Bond formation reactionsusing energy

Category of biochemical bond formation reactions. All require an energy source.

COO-

C

C

C

COO-

H

COOH

H

HO

H

H

COO-

C

C

C

COO-

O

COO-

H

H

H

COO-

C

C

C

COO-

O

H

H

H

H

+ CO2

NAD+ NADH + H+

isocitrate oxalosuccinate -ketoglutarate

DHase

spontaneous

37

Concepts of bioenergeticsConcepts of bioenergetics

Standard free energy change - Standard free energy change - GGoo

The energy change occurring when a reaction, under standard conditions, proceeds from start to equilibrium.

EquilibriumEquilibriumA + B C + D

K’eq =[ C ] [ D ]

[ A ] [ B ]

38

Standard free energy changesStandard free energy changes

Go can be related to the equilibrium expression by:

GGo’ o’ = -2.303 RT log K’= -2.303 RT log K’eqeq

whereGo’ standard free energy change

R gas constant, 8.316 J/mol T temperature, kelvinK’eq equilibrium constant

39


• 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


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 GGo’o’


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



K’eq = 0.67 M / 1.33 M

= 0.50

Go’ = (-2.303)(8.315 J/mol)(298 K) log(0.5)

= +1718 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 ATPEnergy 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 ATPEnergy from ATP

Using a coupled reaction, it is possible to measure the Go’ for ATP.

Go’ kJ/mol

glucose + ATP glucose-6-phosphate + ADP -16.7glucose-6-phosphate + H2O glucose + Pi -13.8

Sum: ATP + H2O ADP + Pi -30.5

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



Whole organisms.Whole organisms.One can introduce radiolabeled materials and measure any labeled waste products.

Tissue slices and cells.Tissue slices and cells.These have been used to uncover metabolic details. The citric acid cycle was characterized using this approach.

Cell-free extracts.Cell-free extracts.Cells are homogenized in a buffer to release cell components for study.

Documents

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