Bio 2171

Overview of fuel metabolism and building blocks: 19/2/14

Principle remember – P

Outline remember – O

Detail remember – D

Food Composition

- All food we eat contains proteins, carbohydrates and fats in different amounts. E.g. cereals

contain more carbohydrates, meat contains more protein.

- Also contain small amounts of vitamins and minerals.

- The macronutrients (protein, carbohydrates and fat) can all be used as fuels or as building

blocks

Metabolic strategies (I)

- Fuels – macronutrients.

- Xenobiotics – toxic things. Cannot avoid eating them as all plants produce toxins to avoid

being eaten. E.g. alkaloids – but we can eat domesticated forms of these plants that have

less toxins, but they still have some.

- First step – digestion and breaking down into monomers, and then transport into the cell

o Then they can either be used to make new body components

o Or break them down and use them as energy. Can also store them as fuels in

between.

- Lastly use oxygen to break these fuels down and generate energy from it. Oxygen is released

in the form of carbon dioxide and water. Allows us to keep going, warm, and is the reason

we breathe.

- Also some fats, carbohydrates and proteins will be used as building blocks in biosynthetic

pathways to make new body components. Some demands to renew organism i.e. skin or to

grow – important for a foetus.

- Whatever is left is released as waste products.

Metabolic strategies (II)

- Organic matter contains a lot of energy – e.g. can burn wood to generate lots of heat.

- However, in organism we do not wish to release all the energy that is generated as heat –

would just burn.

- Metabolism allows us to withdraw energy from organic matter without ‘burning’. The end

point however, is still the same (carbon dioxide and water).

o This is done by taking electrons out of the molecule in small steps (oxidising NAD+ to

NADH) and breaking it down into smaller components – e.g. break down to acetyl

CoA and release carbon dioxide in TCA cycle. Electrons used to generate ATP.

o ATP is energy currency in the body (carbohydrates, lipids and proteins are oxidised

to produce ATP, though a small amount of heat is generated).

o It is used for all the other things that we need to do: active ion transport – important

in the brain (basis of thinking), maintaining body temperature (thermogenesis),

biosynthesis and muscle contraction.

- Energy efficiency is important

- Electrons are stored via oxidising or reducing something – NAD+ is reduced to NADH and is

the electron carrier in the body (stores electrons). ATP is used to store energy.

Metabolic strategies (III)

- Cellular compartmentalisation

o Breakdown occurs in the mitochondria. When oxidising to generate energy, uses

NADH. Largely generates ATP

o Biosytneshsis (lipids, cholesterol, amino acisd and derivates, proteins) occurs in the

cytosol. Synthesis uses NADPH. Largely uses ATP

Overview of metabolism

How to make a cell (building blocks)

- What molecules do we need to make a cell?

- Only need a small handful of ‘building blocks’

- Though need to make a membrane, and membranes around organelles.

- Need to Make DNA, RNA, enzymes and proteins

- Need all these to make a new cell

Membranes

- Made up of phospholipid bilayer.

- Require phospholipids.

- These have polar head group (Ethanolamine, Serine, have phosphate – amino

acid/carbohydrate derivatives) and non-polar fatty acid tails held together by glycerol.

Building blocks for membranes – fatty acids

- Nomenclature – omega three means that there is a double bond three carbons from the end

(not the acid side).

- Not all fatty acids can be synthesised – need to get some of them from nutrition

- More double bonds means lower melting temperature. Steric acid is solid at room

temperature, whereas olive oil is not (has more double bonds).

- Cis fatty acids are found in nature, while trans are not. Trans fatty acids are considered not

very good – associated with diabetes, arterial sclerosis. They are generated during industrial

processing of fats.

Building blocks for membranes – cholesterol

- Cholesterol is an essential component of cell membranes. However high concentrations in

blood are associated with heart disease.

- Derived from fats and lipids

Proteins

- Organisms are mostly proteins – is responsible for giving them shape and form. E.g. hair,

nails.

- Come in different shapes and forms and do lots of different things. Have structural and

functional forms e.g. haemoglobin.

Building blocks for proteins

- All proteins are made from the 20 amino acids.

- Sometimes however these amino acids have to be modified to make the protein

- Grouped into different kinds

o Non polar aliphatic – have sidechains that look like small fatty acids

o Aromatic – have aromatic sidechains

o Polar uncharged – have hydrophilic groups

o Sulphur containing – have sulphur

o Charged amino acids – either have extra positive or negative charge in side chain

- All have same stereochemical configuration (L) and an alpha amino group and alpha carboxyl

group. Some bacteria have other stereochemical configuration.

Titration of amino acids

- At acidic pH, protonate carboxyl group and amino groups (+ve charge).

- As solution becomes more alkaline the carboxyl group is deprotonated as it is quite acidic.

- Eventually, R group may be deprotonated and finally the amino group loses its proton and at

basic Ph, the amino acid has negative charge.

- The alpha amino is always deprotonated about pH 9 (pKa = 9) and the alpha carboxyl group

is usually deprotonated at pH 2 (pKa = 2). However if there are more than one carboxyl

group the pKa will be higher.

- This is very important in enzyme catalysis – charge may or may not be required (as amino

acids vary with pH)

Nucleic Acids

- Whole lot of metabolism behind nucleic acids

- Be aware that they exist – but not dealing with it so much.

Building blocks for nucleotides

- Carbohydrate (ribose), phosphate and the nucleic bases

- Can be synthesised from other materials, but all things we can eat will also contain them.

- Can synthesise with amino acids, carbon dioxide and formic acid.

Carbohydrates as building blocks

- Some lipids contain carbohydrates and nucleotides contain carbohydrates.

- Ribose (carbohydrate) used to build ATP.

- Lactose (disaccharide) required to make breast milk

- Contain lots of hydroxyl groups (where the name carbohydrate comes from)

Carbohydrates are important

- Chondroilin 6-sulfate – found in bone

- Heparin – found in blood

- Hyaluronate – found in cartilage

- Keratan sulphate – found in hair

- Dermatan sulphate – found in skin

o All are polymers of glucose which have been slightly modified (added sulphate) so

that they can bind water and make complex meshes and networks – provide skin

with elasticity.

- Skin is mesh of proteins and carbohydrates that produce structure that binds water, is tear

resistant etc.

Tiny bit of chemistry

Summary

- Food provides the body with energy and building blocks

- Energy is provided by oxidation of nutrient, particularly glucose and fatty acids

- Energy is store as ATP and NADH

- Carbohydrates, fatting acids, AA also form building blocks of all cells.

- The framework of organic chemistry explains the reactivity of biochemical compounds

Carbohydrates and Energy (24/2/14)

Carbohydrates are everywhere – sugars, starch

Carbohydrates in nutrition

- Sucrose – found in fruits. Broken down into sucrose and fructose – sweet

- Lactose – not so sweet, found in milk

- Amylose – contained in starch, subunit. Alpha -1,4 glycosidic bond (O atom points down).

Can digest easily

- Amylopectin – branch in starch. Alpha -1,6- bond. Very frequently used in food industry –

makes food less liquid (solidifies it).

- Cellulose – Beta -1,4- bond (O atom points up). Cannot digest – very small difference in the

conformation of a molecule can change whether it is digestible or not.

Cyclic and linear forms of glucose

- Most glucose present in circular form – < 95%. Other >5% in linear form. Different forms are

in equilibrium.

- In linear form glucose is an aldehyde, which is a reactive molecule due to the partial positive

on the carbon. Means that groups with a free electron pair can add to the aldehyde.

Advanced glyction end products (AGE’s)

- This causes a problem in diabetes, where people have a higher than normal blood glucose

concentration.

- This means that there will be more than normal glucose in the linear chain form – with an

exposed aldehyde group.

- This means there will be more advanced glycation end products (AGE’s) being formed, as the

aldehyde in glucose can react with the amino group in an amino acid (e.g. to an amino acid

in Haemoglobin) to produce a Schiff base which creates a glycated haemoglobin – called an

AGE which can lead to protein cross linking.

- The measure of glycated haemoglobin is an indicator for the severity of diabetes in people,

and they are indicated to cause some of the side affects of diabetes – such as glucose

reacting with blood vessels of cells and cause vascular problems i.e. vascular sclerosis and

other things – cataracts.

- So high blood glucose can cause damage to cells over long periods of times – uncontrolled

levels of glucose in the blood is bad, as it will eventually result in these problems. (glucose is

a reactive molecule and can cause protein modification if the concentration is sufficiently

high).

Overview of energy production from glucose

- Energy is made in biological systems is via glycolysis, the TCA and then the electron transport

chain – energy is made by extracting electrons.

- Overall energy of the reaction of burning glucose can be obtained using a bomb calorimeter

(2800kJ/mol)

- In biological system burning is not useful – want ATP and NADH (takes out electrons) as we

can do work with it.

- Want to get the energy out in bits and pieces (this is what these two molecules do).

Major questions:

What is the role of ATP?

What is the role of NADH?

Tim tams slide?

- Energy required is dependant upon what activity a person is undertaking?

Energy flow in chemical reactions

- If ΔG is negative – will accumulate products – backwards reaction will not be spontaneous. If

ΔG is positive, will accumulate reactants – forward reaction will not be spontaneous.

- If ΔG is zero, reaction will be at equilibrium

- ΔG – any change of free energy

- ΔG0 – change of free energy starting with concentrations of 1M.

- ΔG0’ – change of free energy under biochemical conditions (250C, pH 7.0, water 55.5M).

Free energy and equilibrium (I understand this right?)

- If we have more products than substrate at equilibrium (high equilibrium constant), than will

have greater free energy (maxiumum amount of work that can be obtained from the

reaction).

- Equilibrium is directly correlated to the amount of energy that can be made in a biochemical

system.

Thermodynamics vs. kinetics

- The ΔG represents the amount of energy that can be made from the reaction. It is the net

energy change in the reaction.

- The ΔG does not say anything about the speed of a reaction – the activation energy

determines the speed.

- The activation energy must be overcome for the reaction to proceed

- E.g. Tim tams have lots of energy, but it does not combust spontaneously as its compounds

are quite stable – requires an enzyme to reduce the activation energy to break it down.

Energy flow in chemical reactions

- Lactose + H2O -> glucose + galactose ΔG0’ = -15.9 kJ/mol

- This reaction gives us energy because the concentration of water is 55.5M, whereas the

concentration of metabolites is only 1-5mM – and so the equilibrium of the reaction is

pushed towards the right.

- So by definition, hydrolysis will always have a –ve free energy due to the involvement of

water (will generate energy).

- Conversely, when water is generated ΔG>0, as it is pushing it against equilibrium.

- The other reaction that always produces lots of energy is oxidation – so long as it is more

reduced than carbon dioxide or water, energy will be able to be generated from it.

- Fats are a good example – carbon has all its electrons, and so these can be oxidised away via

reducing oxygen, because it is such a good electron acceptor.

Storing energy in ATP

- Hydrolysing ATP produces a large amount of energy.

- However, the energy produced via hydrolysing ATP is conserved by keeping it a large degree

out of equilibrium (If reaction is in equib – ΔG = 0 and no energy can be obtained).

- ATP also generates energy because of the presence of adjacent negatively charged

phosphates, and this in addition to the concentration of water allows it to produce lots of

energy via hydrolysis.

o If ATP -> ADP + Pi was allowed to run to equilibrium, there would be 1 ATP to

100,000 ADP (0.00001).

o As this would be equilibrium, no energy would be obtainable.

o In normal cells, the ratio is from 2.5 to 9 ATP: 1 ADP (which is massively out of

equilibrium).

o i.e. we are charging the battery to the maximum possible amount, even though

there are lots of processes that use ATP.

o If the ATP ratio falls even a small amount, the cell would die – equilibrium needs to

be multiple tenthousandths away from the substrate.

o Most nutrients we eat are used by the body to ensure that this reaction is kept far

out of equilibrium.

Using ATP to drive unfavourable reactions

- Conversion of glutamic acid to glutamine usually does not occur (ΔG = +50ish kj/mol).

- However, can take a detour via phosphorylating with ATP to produce a high energy

intermediate, can change free energy to ΔG = -18kJ/mol. (free energies of linked reactions

can be added and subtracted, and if it is favourable, than the overall reaction can proceed).

For what do we use energy?

- The mitochondria uses most of the ATP in the cell, and these are coupled to ATP synthesis

(protein synthesis both in cytosol and mitochondria, Na+Ka+/Ca+ - ATPase synthesis – crucial

for maintaining membrane potential, myosin ATPase (muscle work), Gluconeogenesis and

Urea synthesis etc.)

- This makes sense as the mitochondria is where most of the ATP is produced

Storing electrons (oxidation)

- One of storing energy is keeping it out of equilibrium – e.g. ATP stores chemical energy in

this way.

- Another way is to conduct redox reactions. Redox reactions are different from hydrolysis as

there is a net exchange/production of electrons.

- We need something else other than ATP to store electrons from redox reactions – so NAD+ is

used. NADP+ has a phosphate group esterified onto one end instead of a hydroxyl.

- The ‘business end’ is a positively charged nitrogen, which acts as an ‘electron sink’.

- It takes up two electrons and a H+ in the process.

- We can see reduced NADH using a spectrophotometer as it has a strong absorption where

NAD+ does not – can tell if cell is working hard or not (via creating more NADH).

- ATP stores chemical energy, NAD+ stores electrons.

- However this is only short term storage, and there is a high turnover of NADH back to NAD+

An example: Ethanol metabolism

- Ethanol can be reduced to Acetaldehyde to produce NADH – captures the electrons.

- This happens first in liver, and muscle oxidises it further to acetate to produce another

molecule of NADH.

Summary

- The free energy of a reaction is determined in part by its dislocation from the equilibrium

concentration.

- Biological reactions take place in aqueous environment; water is present at a concentration

of 55.5M driving hydrolysis reactions.

- The hydrolysis of ATP is kept far from equilibrium in living cells. ATP hydrolysis at equilibrium

indicates a dead cell.

- Combining the out of equilibrium reaction of ATP with other reactions can drive otherwise

unfavourable reactions

- Electrons from oxidation reactions are stored as NADH + H+

- Oxidation is the removal of electrons. In biological oxidations oxygen is most of the time the

ultimate electron acceptor forming O2.

- The strong affinity of oxygen for electrons explains why oxidation is usually exergonic

- In biological systems electron carriers are used as an intermediate storage of electrons

(NAD, FAD)

Digestion and absorption of carbohydrates (26/2/14)

- Absorb glucose in the intestine after a meal

- Then store some carbohydrates in liver as glycogen – any excess that cannot be stored in

glycogen will be turned into fat and stored in adipose tissue.

- Brain tends to use glucose straight away – and usage is constant

- Will also build up glycogen in muscle, which need to be replenished after exercise

- All these processes are governed by Insulin, which is secreted by the pancreas.

Digestion of carbohydrates

- Begin digesting in mouth by turning things into finer particles (enzyme in saliva alpha-

amylase – digests starch, splits amylose at oxygen linkage to turn it into individual units of

glucose, though there is a different enzyme for breaking down the branched parts).

- Further digested in stomach

- However most of digestion is done in small intestine.

- Once stomach has a meal and starts extending, the pancreas begins to secrete the enzymes

required to break down all the nutrients – i.e. all disaccharides and polymers of glucose are

broken down into monomers.

Digestion of Carbohydrates

- Break multimeric carbohydrates down in the intestine into oligosaccharides and then further

down into the individual monomers and are then absorbed in intestine. E.g. splitting starch

will get maltose and this will then be broken down into glucose and absorbed in intestine. Or

sucrose will be broken down into fructose and glucose etc.

- True for all multimeric nutrients – break down in intestine into individual units (individual

fatty acids for fats, amino acids for proteins, monosaccharides for carbohydrates)

Anatomy of intestine

- Intestine has very large surface area – with invaginations and finger like protrusions called

villi.

- Villi cover whole surface of the intestine, and the finger like complexes are loaded with

digestive enzymes.

o In the membranes of the individual cells are anchored enzymes which break down

carbohydrates as shown in diagram, such as isomaltase.

o They also take up nutrients and release them on the other side of the epithelial cells,

and each finger like villi has blood vessels and lymph vessels which go right to the tip

o This allows nutrients to pass into the blood and lymph system and make their way to

the liver

- Most are called enterocytes, which are involved in this nutrient absorption. Though there

are also other ones called goblet cells which produce mucus that allows food to pass through

the small intestine easier.

Lactose intolerance

- People in regions that historically have not consumed much milk can be intolerant to

lactose, though there are different degrees of lactose intolerance.

- Whats missing is the enzyme that breaks down lactose – lactase

- So lactose passes through intestine without being absorbed, and reaches the large intestine

where there are large amounts of bacteria – these then switch to eating lactose

- The bacteria consuming lactose results in large amounts of lactate being produced

(fermentation), which builds up and increases the osmotic pressure in the intestine

- This results in water flowing into the intestine, and diarroheoura results

Transport of carbohydrates

- Once all carbohydrates have been broken down into monomers, they need to be

transported inside the cell.

- Inside cell membrane there are proteins which facilitate the transport and uptake of sugars

- E.g. move glucose and fructose from one side of the membrane to the other

- Also have transporters which allow sugars to exit into the bloodstream

Glucose-galactose malabsorption

- Rare inherited disorder due to the lack of a functional glucose-galactose transporter –

caused by mutations in SGLT1.

- Life threatening due to bacteria producing so much lactic acid that the water being

withdrawn from the body results in severe life threatening diarrhea

- Fructose not affected

- Treated by strict diet that does not contain starch and other carbohydrates. Can eat protein

and fructose

Indigestible carbohydrates

- Bacteria in intestine are useful

- However they need something to eat – this is the reason why we cannot absorb all

carbohydrates that we eat – need to feed our microfauna. Cannot digest…

o Cellulose

o Pectins

o Agar

Why do we need fibre?

- These indigestible carbohydrates are called fibre.

- This fibre is important for bacterial metabolism (feeding bacteria in gut).

- This generates the fermentation products such as butyrate, acetate etc. and these can reach

high concentrations.

- The cells in the colon actually live solely on these fatty acids, as all other nutrients have

already been absorbed by the time the food reaches them – have the metabolic processes to

digest them. Without fibre, colon would starve.

- These nutrients also are involved in the cell differentiation of cells in the colon, and so

having a low fibre diet can lead to colon cancer.

- These components are then used to drive cell differentiation in the colon.

Why are some carbohydrates indigestible?

- No enzyme to cleave them and break them down into monomers which can be absorbed

- Without an enzyme, would not be able to metabolise anything.

- Have purpose as food for intestinal microflora

Detour: How enzymes work

- Enzymes facilitate a reaction

- Enzyme b) would not function as though it binds the substrate, it doesn’t make it do

anything – would just trap it and in doing so prevent the reaction in a).

- Enzyme c) however fits to the transition state, and in doing so when it interacts with the

substrate it forces it into that state – meaning that it now only requires a small push for the

stick to break.

- This is the secret of catalysis – by having interactions with the transition state of the

substrate, this allows it to more easily undertake the reaction – as shown in the free energy

graph in C.

- Allows biochemical reactions to occur on a reasonable timescale.

How enzymes work, hydrolysing carbohydrates

- Simplification of breaking down carbohydrates, but the same basic principle applies

1. The have a shape such that allows the disaccharide to bind to the enzyme, and

have the shape such that the glycosidic bond is exposed.

2. Asp and Glu are acidic amino acids – Glu is protonated, Asp is not. This is

because within an enzyme the characteristics of the residues can be quite

different to that in solution.

3. Placement of the disaccharide makes the approach of the Asp negatively

charged oxygen easier and dissociates the proton on glutamate.

4. This splits the glycosidic bond

5. We then use hydrolysis to release the glucose molecule and reprotonate the

Glu.

Types of catalysis

- General base

o One example is when NAD+ takes a hydrogen from ethanol to oxidise it

o The B base responsible for this reaction can be any form of free electron pair (as a

free electron pair can form a bond) – Asp residue in previous example acts as a

general base.

- General acid

o As before, when the glu reside had a proton that could dissociate to form hydroxyl

group to hydrolyse the sugar. (step 1)

o Main property is having a proton that can dissociate

- Covalent intermediate

o Where a covalent intermediate is formed between the enzyme and the substrate

o Step three in above diagram

- Metal ion catalysis: Enolase

o Has metal ions that are positively charged, and so they attract electrons.

o This can shift electrons within the molecule and stabilise its transition state –

allowing the reaction to proceed.

Summary enzymes

- Enzymes catalyse chemical reactions, they speed up the reaction

- At the end of the reaction, the enzyme is in the same sate as the beginning

- They do not change the equilibrium of the reaction

- Active site residues react with substrate

- Covalent intermediates may be formed

- Transition state binds tightly to the enzyme

- Enzymes do not change the equilibrium of the reaction or the directionality (free energy tells

us the equilibrium of the reaction, enzymes only determines how fast it goes)

Types of catalysis: general acid or base, covalent catalysis, metal ion catalysis