Lecture 1 Outcomes

1. Function of basic components of Chlamydomonas cells

Nucleus – similar to the nucleus of a human/plant/animal cell

Has a basal body – found at the base of the flagellum – where the microtubules develop

Mitochondria – ATP factory of the cell

Chloroplast

o Inside the chloroplast is a reigion called the pyrenoid

Where carbon fixation takes place

Energy transduction – chloroplast and mitochondria work closely together

Eye spot – orangey in colour because of a pigment called carotenoid

o Found within chloroplast but not directly related to photosynthesis

o Enables the cell the orientate itself in relation to light

2. Relative usefulness of various biological characteristics as measures of complexity

Cell size

o Chlamy is 10 microns in diameter, much bigger than a bacterium

Genome size

o In many cases it isn’t a good indicator – can mislead you

PCG – Protein Coding Genes

o How many proteins do your genes code for

Rise of Multicellularity

o Volvox – 50 000 cells in one organism

Somatic cells on the outside and reproductive cells on the centre

3. Advantages to Chlamydomonas in being phototactic

Chlamy uses its eyespot for phototactics - the movement towards or away from light

It wants to move towards light because they want to harvest photons for

photosynthesis

4. Reasons why Chlamydomonas might move AWAY from a light source

Running away from bright light suggests light may destroy the photosynthetic apparatus

by creating free radicals (ROS – reactive oxygen species)

The idea that light can be harmful even if it’s in the

visible spectrum

5. Basic structure of rods and cones as photoreceptor cells

There are two types of photoreceptor cells, rods and

cones

o They are modified neurons

o They sit on the retina

The rod is made up of disks which has many individual

photoreceptors

o Blue dots on the disks

o It is the photoreceptor that tracks the light

6. Major components involved in phototransduction and their role

Photon changes the shape of the photoreceptor from cis-retinal to trans-retinal and

activates a pathway called phototransduction

o Activates a protein called transducing which activates an enzyme called

phosphodiesterase

The sodium transporter is on the plasma membrane of the photoreceptor cell

The sodium pump is regulated by cyclic GMP

o When cGMP is bound, sodium is transported into the cell – sodium influx

cGMP is bonded to the 3’ and 5’ position and the G protein activates phosphodiesterase

which breaks the bond in cyclic GMP

phosphodiesterase cleaves the 3’ end so it is only bound on the 5’ end which generates

5’-GMP and the consequence of that is the transporter turns off and shuts off the

sodium pump

o the consequence of that is the membrane hyper polarizes – the voltage across it

becomes even greater

in the light the membrane hyper polarizes which leads to an electrical

signal being sent down the membrane surrounding the photoreceptor

cell

200 million bits per second move along the optic nerve to the brain

Lecture 2 Outcomes

1. Relationship between excited states of a pigment and its absorption, fluorescence emission

spectra

When a pigment absorbs a photon of light, an electron in the pigment is excited from

the ground state

If it absorbs a higher energy photon of light (ie. Blue) it will be excited to the higher

excited state, but will decay very quickly to the lower excited state

If it absorbs a lower energy photon (ie. Red) it will be excited directly to the lower

excited state

When the electron falls from the lower excited state some energy is lost as heat, but the

rest through fluorescence

o The fluorescence will be a longer wavelength that the original light because

some energy has been lost to heat

o The longer wavelength will cause the colour to be slightly different than the

original photon absorbed

2. Region of the electromagnetic spectrum known as “visible light”

The most abundant form of light on the surface of the Earth and is the light that can be

detected by the human eye (ROYGBIV)

Typically absorbed and emitted by electrons in molecules and atoms that move from

one energy level to another

This action allows the chemical mechanisms that underlie human vision and plan

photosynthesis

3. Relationship between wavelength and energy content of a photon

The wavelength and the energy of a photon are inversely proportional

o The shorter the wavelength, the higher the energy

Gamma rays have short wavelength, radio waves have long wavelength

4. Molecular characteristic of visible pigments that make them able to absorb light

Molecules that absorb light are called pigments

They have a conjugated system

o Alternation between a double and a single bond

It indicates a certain kind of electron configuration: non-bonding Pi orbital electrons

o It is those electrons that are going to interact with the photons of light

An exception is retinal – it does have a role in bonding

5. Relationship between pigments and associated protein

Whether it is chlorophyll or retinal, these pigments are not free, they are bound very

specifically to proteins

If proteins and pigments are bound, when you isolate the protein you can actually keep

the pigment attached

o It is bound non-covalently to the protein

If you isolate mitochondrial proteins, you don’t see anything when you put them

through the gel electrophoresis – they don’t have any pigment so you have to stain the

gel

6. Four “fates” of the excited state of chlorophyll resulting from absorption of photons

Can lose it all as heat – the higher state can simply decay and you lose the energy

o In Chlamy, if you lost all the energy to heat the

cell would die

Can lose a little bit of energy to heat, and go to a sub-lower energy state, then lose the

rest of the energy as fluorescence

o Since you lost of little bit of energy to heat, this is a different colour red –

fluorescence has a slightly longer wavelength

You can do work with the energy – use it in photochemistry

o Use the energy to change the structure of the pigment

You can transfer the energy to a neighbouring pigment

7. Reason(s) why relative fluorescence is different in isolated chlorophyll vs. intact cells when

exposed to light

If the chlorophyll is intact with the cells, the energy that is absorbed from exciting the

electrons is used to power photosynthesis, causing no fluorescence to occur because

the energy is already used up

When the chlorophyll is isolated, the excited electrons have to fall back to their ground

state which explains why fluorescence is much higher

o This does not happen in chloroplasts since the excited electron is passed to the

electron transport chain and the energy used to create a H+ gradient which

drives ATP synthesis

8. What accounts for the fact that chlorophyll is green in colour

There is no green excited state

o There is no excited state between the red and the blue excited states

Green photons are just lost – either reflected or transmitted through that pigment

9. Quantitative relationship between photons and excited electrons

When you have an electron and a photon, one photon can excite one electron – one to

one equivalency

10. Relationship between energy of photon and energy required to excite electrons in order for

photons to be absorbed

The energy in the photon has to exactly match the energy difference between the

ground state and the excited state

11. General structure of photosystem

Comprised of 2 parts

o Purple antenna – on the outside: protein, chlorophyll individually bound to the

protein

o The reaction centre –in the middle

No photochemistry in the antenna, only phototransfer

o Energy from one chlorophyll is transferred one to another

o Not moving the energy, just moving the excited state

When the energy reaches the chlorophyll in the reaction centre the

oxidation of chlorophyll occurs, releasing an electron, this drives

electron transport in the thylakoid membranes of chloroplast as well

as bacteria

12. Similarities and differences of the light capturing and photochemistry of phototransduction

(retinal) vs. photosynthesis (chlorophyll)

The photochemical event in the eye (retinal) being the cis/trans change in conformation

and in a photosystem the event being the oxidation of chlorophyll

In phototransduction (retinal), photochemistry takes place in photoreceptor located on

the disks

o The actual chemical event is the isomerization of retinal

o Retinal needs to be changed in shape(conformational change), but in order to

do this it needs energy

o It gets the energy from capturing photons of light (photoisomerization)

o It interacts with bonding electrons

In photosynthesis (chlorophyll) there is no photochemistry in the antenna, you are not

changing the molecule, there is simply energy transfer

o Energy shifts from an excited chlorophyll to a neighbouring one, original

chlorophyll goes back to the ground state and the neighbouring pigment gets

excited

o Pigments must be very close together

o Photochemistry takes place when the energy reaches the reaction centre

o In the reaction centre, reaction centre chlorophyll is oxidized

o Chlorophyll gets to an excited state, and an electron is pulled off

Electron drives electron transport

o Interacts with nonbonding electrons

13. How are excited states of antennae pigments organized to provide for energy transfer to

reaction center

The chlorophyll are close enough that energy from one chlorophyll is transferred from

one to another

When the energy reaches the chlorophyll in the

reaction centre, the oxidation of chlorophyll occurs,

releasing an electron

This electron drives electron transport in the

thylakoid membranes of chloroplast as well as

bacteria

The pigments ground states would be lined up with

the excited states of the neighbouring pigment such

that the pigment farthest from the reaction center

will have the highest excited state and the one with

the lowest excited state will be the closest

14. Structure of rhodopsin

Consists of the protein opsin which is covalently bound to the cofactor retinal

Each outer segment disc contains thousands of visual pigment molecules

About half the opsin is within the lipid bilayer

15. Effect of photon absorption by 11-cis retinal on retinal structure followed by association with

opsin protein followed by interaction of transducin with opsin

Retinal is the reddish-purple part (it absorbs green-blue colour) which is surrounded by

the protein opsin

o Opsin binds retinal

Cis – hydrogens on the same side of the double bond

Trans – hydrogens on opposite sides of the double bond

Double bond prevents the molecule from freely rotating

o Sigma bond and pi bond make up the double bond

The bond breaks – a photon of light is captured, one of the electrons in the carbon-

carbon double bond is excited and breaks the bond to rotate it, and then the bond

reforms

o Goes from 11-cis-retinal to all-trans-retinal

o This is called photoisomerization

o The photochemical event that occurs in the photoreceptor

Transducin needs to interact with the opsin protein

o There is a clef in opsin that allows it to interact and bind

o Single transduction pathway leading to vision for one photon becomes active

In the dark when you have 11-cis retinal transducing can’t get in there, there is no clef,

so it cannot bind

Upon photon absorption the 11-cis retinal shifts to the trans configuration and can no

longer bind so the pigment detaches from the opsin – this does not happen in a

photosystem

o In a photosystem the chlorophyll’s always stay put

This opsin can’t absorb any light anymore so this opsin has to be recycled and a new cis

retinal has to be binded

TAKE HOME: ** in the transconfiguration the molecule is simply lost from the opsin,

and this doesn’t occur in the photosystem

TAKE HOME: ** the shift from cis to trans changes the shape of the protein (opsin),

opening up a clef so now transducing can interact

o Once it interacts it activates phosphodiesterase, which hydrolyzes cyclic GMP

and the Na+ channels close, hyperpolarizing the membrane, and sending an

electrical impulse to the brain

16. Reasons why life has evolved to detect the narrow band of energy represented by “visible

light”

Visible light is abundant on earth

It is energetically perfect for absorption without obliterating the molecule

Lecture 3 Outcomes

1. Reasons why photosystems have antenna proteins while the eye doesn’t

If you are photosynthetic you want to harvest as much light as you can, whereas in a

photoreceptor the arrangement of rods and cones are trying to convey information and

create a picture so too much light would be counter productive

2. Points of control for regulation of protein abundance

Control transcription – the DNA to mRNA synthesis

o The transcript is the product of transcription

o The transcript is the mRNA

Control translation – control the conversion of mRNA to protein

Decay of mRNA – how long does the mRNA stick around

3. Factors affecting mRNA transcript abundance

If you are transcribing lots of DNA, you should make lots of transcript, but that’s not

necessarily the case

There is another step called mRNA decay/turnover

o When mRNA is made, it doesn’t stick around forever, some breakdown after 20

mins, a few hours, etc.

4. Steps in making a Northern Blot for measuring mRNA transcript abundance

Isolate RNA from cell or tissue samples

Quantify how much total RNA you have, and load the same amount in every lane

Gel electrophoresis

You don’t see any mRNA on the gel because their abundance is much lower

Transfer all the RNA to a membrane so you can probe the membrane and detect a

specific RNA – radioactive gene-specific “probe”

o Most important part

Can make single stranded DNA (radioactive) probe that would hybridize to the mRNA

that corresponds to its complementary sequence (this is the radioactive gene-specific

probe)

5. Relative abundance of various types of RNA in typical cells

97% of all RNA is ribosomal RNA

o 2/3 of a ribosome is RNA

Only about 3% is mRNA

Prokaryotic ribosome is different that eukaryotic ribosome

6. Steps in making a Western Blot for measuring protein abundance

Use gel electrophoresis to separate native proteins by the length of the polypeptide

SDS-PAGE gel is then transferred to a membrane

It is stained with antibodies (raised in rabbit/chicken/goat)

Can detect the abundance of protein in the blot

7. Characteristics of constitutive vs. induced vs. repressed gene expression kinetics

Biological system don’t like too much heat

3 types of kinetics that are possible:

o Constitutive expression - actin

No response to a change in temperature

o Induced expression - HSP

Abundance goes up

o Repressed expression

Abundance goes down

8. Varieties of defects that might account for lower levels of functional photoreceptors

Transcription is shut down or translation is shut down – defect

mRNA could be decaying too quickly – never accumulate much transcript

may be a defect in a transcription factor

** Mutation to the opsin gene – when transcription occurs it makes mRNA but makes

the wrong protein so it doesn’t fold correctly

May absorb too much light – damaging to the photoreceptor

Opsin could be perfectly fine, rhodopsin requires retinal in order to work, so there may

be no defect in opsin, but a defect in retinal biosynthesis

9. Relationship among polypeptide, apoprotein, cofactor and functional protein

Retinal is NOT coded by a gene

o It is a product of a biosynthetic pathway

o The pathway is catalyzed by enzymes

o It is not a protein, it is a cofactor

The cofactor and the apoprotein (the protein before it accepts the cofactor – opsin isn’t

functional until it binds to the cofactor) come together to form a functional protein

o All pigment protein processes would have to go through this

This is what we call post-translational modification

Not all proteins need this cofactor, after translation they are fine – don’t need any post

translational modifications

10. Relationship between protein folding and function

For a protein to be functional it HAS to fold

o Acquire a correct three dimensional shape (confirmation)

Polypeptide (chain of amino acids) hasn’t folded correctly yet, once it has then you have

a functional protein

11. Factors affecting proper protein folding (Afensen’s dogma)

If you have an enzyme that has the correct tertiary shape you can measure the product

of the reaction by measuring the colour change in the test tube

o Native (tertiary) is 100% active – colourful

You can add Urea – disrupt the bonding arrangements which causes it to unfold

(denature)

o No colour when experiment is conducted again

He was able to show that the protein will refold once the Urea is removed

o Native >90% active

Protein folding is spontaneous, in milliseconds

o Doesn’t require energy to happen – don’t need to add ATP

o Dependent solely on primary sequence

The order of the amino acids is the only thing that dictates the protein

Lecture 4 Outcomes

1. Meaning of:

Potential Energy o Energy that is stored

Kinetic Energy o Energy that a body possesses by being in motion

Chemical energy o Energy that can be released by a chemical reaction

Closed System o Exchanges energy but not matter with the surroundings

Open System o Exchanges and energy and matter with surroundings

Isolated system o Doesn’t exchange matter or energy

First Law of Thermodynamics o Energy can be transformed from one form into another or transferred from

one place to another, but it cannot be created or destroyed

Second Law of Thermodynamics o The total disorder of a system and its surroundings always increase

Entropy o The measure of disorder or “randomness” in the universe

Spontaneous reaction o A reaction that occurs without needing to be driven by an outside source of

energy

Enthalpy (H) o Potential energy that molecules have

ΔH o Change in enthalpy

Exothermic o Reactants have more potential energy than the products (-ΔH)

Endothermic

o Products have more potential energy than the reactants (+ΔH)

Gibbs Free Energy o Energy that is available to do work

Exergonic o Less disordered (-ΔS)

Endergonic o More disordered (+ΔS)

ΔG o Change in free energy

Catalyst o A substance that increases the rate of a chemical reaction without itself

undergoing any permanent chemical change

Rate of reaction o How fast or slow a reaction proceeds

Energy of activation (EA) o The amount of energy required to reach the transition state

Transition state o The higher energy state the reactants need to reach in order for the

reaction to proceed Reach it by hitting other molecules or gaining energy from the

environment

Kinetic stability o A molecule or compound is kinetically stable when it requires a lot of energy

to reach the transition state

Active site o The region on an enzyme that binds to a protein or other substance during

the reaction

Catalytic cycle o The complete sequence of steps by which a chemical transformation is

accelerated in the presence of a catalyst 2. Why life does not go against the second law

Some people say life disobeys the second law because it is highly ordered

We maintain low entropy because of our huge amount of energy input 3. Why life needs to consume energy

Need to consume energy in order to keep low entropy o The energy we consume is in our food

4. Components of Gibbs Free Energy equation

ΔG = ΔH -T ΔS

Free energy is dependent on two things o Enthalpy – potential energy the molecules have

+ Endothermic (product has more potential energy) - Exothermic (reactant has more potential energy)

o Entropy – the amount of disorder + More disordered (endergonic) - Less disordered (exergonic)

5. Whether or not a given reaction will be spontaneous, given ΔG

Reactions tend to be spontaneous (-ΔG) when the products have less potential energy than the reactants

When products are more disordered from the reactants – entropy is higher in the products than the reactants

For a reaction to proceed spontaneously, ΔG needs to be negative 6. Role of enzymes in endergonic vs. exergonic reactions

A -----> B -----> C

A to B is spontaneous (usually –ΔH), but an enzyme can make it faster

B to C is nonspontaneous (usually +ΔH), adding an enzyme doesn’t get you

anywhere

o An enzyme cannot change the sign of the free energy change – cannot

convert +ΔG to a - ΔG

o Only way to get this reaction to proceed is to add energy

o In a cell this energy usually comes from ATP and there is an enzyme that

goes with this

An enzyme can ONLY catalyze reactions that are spontaneous

7. Relationship between activation energy and rate of reaction

Amount of energy required to reach the transition state is called the activation energy EA o At the transition state the bonds are strained and ready to break

If you don’t need very much energy to reach the transition state then the reaction can occur very quickly

If you need a lot the reaction can take a very long time 8. How enzymes increase rate of chemical reactions

They lower the activation energy so more molecules can reach the transition state o Simply change the path the reaction takes

An enzyme is just a protein

The red and blue substrates must come together in this formation to reach the transition state – it’s usually rare they get this orientation but the enzyme mimics the orientation they need to reach the transition state

The substrate molecule needs a certain charge across it and it’s usually rare but the amino acids that make the active site of the enzyme provide that charge

The substrate may need to be strained – conformation strain

The Catalytic site mimics the transition state **** 9. Why biological systems need enzymes

Low temperature – the question that evolution was faced – how do you get reactions to proceed more quickly without having to raise the temperature?

o Biological molecules cannot handle high temperatures or high pressures

o The Earth is cold, to get reactions to proceed quickly you need enzymes 10. Importance of tertiary structure to enzyme function

When a substrate molecule gets close to the enzyme it causes a change in the shape of the enzyme

o This is called induced fit

The enzyme is a protein, it requires the correct tertiary conformation – 3D structure of the enzyme is vitally important

o Bonding arrangement that gives rise to tertiary structure have some flex because the shape of the protein changes upon substrate binding

11. Link between enzyme function and growth rate

The temperature at which the enzyme function is the highest will be when the growth rate is the highest

Rate of motion increases with temperature

When the temperature is too high though it will denature the enzyme and have no growth rate

12. How tertiary structure bonding arrangements are different depending upon the temperature habitat of the organism

Depending on the temperature the structures bonding arrangements are different because the enzyme is most active at some temperatures (ideal)

When the temperature gets a little too high, the bonds will start to break denaturing the enzymes structure

Extremophiles

Lecture 5 Outcomes

1. Meaning of:

Hydrophilic o Having a strong affinity for water, dissolves in water - polar

Hydrophobic o Lacking an affinity for water, doesn’t dissolve in water – nonpolar

Fatty acid o Carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated

Saturated o Containing the largest possible amount of a particular solute o More saturated = more fluid

Membrane fluidity o Viscosity of the lipid bilayer in a cell

Hydrogenation o Adding hydrogen molecules to unsaturated fats

Desaturase o An enzyme that removes two hydrogen atoms from an organic compound, creating a

carbon-carbon double bond

Membrane permeability o How permeable a membrane is to letting certain molecules through

Transmembrane protein o A protein that spans the entire biological membrane

Simple diffusion o Using the concentration gradient to move small molecules across a membrane

Facilitated diffusion o A channel in the membrane that doesn’t interact with the hydrophobic core, but allows

molecules to pass through

Active transport o Going from a region of low concentration to high concentration – against the gradient

“ATP-Binding Cassette” (ABC) transporter o Function is to transport specific molecules in or out of the cell

Cystic fibrosis o A disease that causes death and a buildup of mucus because they are missing a form of

proteins

Cystic Fibrosis Transconductance Regulator (CFTR) o Keeps the lining of your lungs wet by pumping chlorine ions

∆F508 o Deletion of phynelalonine at location 508

Chaperone protein o Help to detect for proteins that are folded improperly

“ER quality control” o Looks for proteins that are not folded properly and attacks them

Proteasomes o Very large protein complexes inside all eukaryotes and archaea

Proteases o An enzyme that breaks down proteins and peptides

2. Role of fatty acids in membrane structure

Membranes can be more than 50% protein o The backbone of the membrane is a lipid bilayer, but there are lots of proteins

embedded or interacting with the membrane

The major membrane lipid is what we call a phospholipid o This is the fatty acid part of the lipid, there are 2 fatty acid tails that are hydrophobic o They can either be saturated or unsaturated

3. Relationship of fatty acid saturation levels on membrane fluidity

If you have fatty acids that are made of just saturated fatty acids, they pack closer together and the membrane is less fluid

The more unsaturation you have, the more fluid your membrane will be at any given temperature

4. Relationship of temperature on membrane fluidity

Desaturase is an enzyme that creates unsaturated fatty acids

When the temperature is low there is a high abundance of desaturase because it helps the membrane become more fluid

As the temperature increases the abundance of desaturase does down so that the membrane (that is already becoming more fluid from the heat) doesn’t become even more fluid

The organism is attempting to maintain membrane fluidity within a very narrow range 5. Relationship of fluidity to membrane functions such as transport

The more fluid that a membrane is the more types of molecules will be able to transport through the membrane

If the membrane is not fluid enough then simple molecules won’t be able to diffuse through the membrane

6. Properties of saturated vs. unsaturated fats

Saturated: has a lot of hydrogens, very linear molecules o If you have fatty acids that are made of just saturated fatty acids, they pack closer

together and the membrane is less fluid

Unsaturated: less hydrogens, contains kinks where there are carbon-carbon double bonds o The more unsaturation you have, the more fluid your membrane will be at any given

time 7. Role of desaturases in fatty acid biosynthesis

Desaturases are the group of enzymes that act on saturated fatty acids and introduce double bonds, creating unsaturated fatty acids and thus kinks in the backbone

8. Relationship of bacterial desaturase expression vs. temperature

The lower the temperature the higher the abundance of desaturase

As temperature increases the levels of desaturase goes down 9. Role of size and charge in movement of molecules across biological membranes

If you are small and uncharged, you can move right through

o Oxygen, carbon dioxide – diffuse down a concentration gradient

As you get bigger or charged, it becomes difficult to diffuse through o Glucose, chloride need to be transported across

10. Characteristics of transmembrane proteins that enable them to interact with hydrophobic core of membrane

Inside of the core which is interacting with ions and charged particles is hydrophilic, the outside

which is interacting with the fatty acids of the membrane are hydrophobic

There tends to be 2 trends that you see in membrane proteins o They have alpha helices

The hydrogen bonding that gives rise to this structure minimizes the charges of the protein backbone so it can interact with the fatty acid tails

o The primary sequence of a protein does not provide you with information of where the active site is

Membrane proteins do have a signature in their primary sequence though

the part of the protein that interacts with the membrane tends to be made up of nonpolar amino acids

17-20 amino acids make up the transmembrane spanning domain Can detect if it is a transmembrane protein based on primary sequence

11. Factors influencing simple & facilitated diffusion

Diffusion o Down a concentration gradient o High concentration to low concentration o What drives this diffusion is the free energy change

More free energy when you have a difference across a membrane

Facilitated Diffusion o Uses some kind of protein pore o Movement is still diffusion based, but it cannot move through the hydrophobic core so it

needs to be facilitated through o Pores are very specific

12. Transport against a concentration gradient (active transport)

ABC Transporter o Active transport o Pump things against a concentration gradient

Low to high concentration o Against the free energy change – need energy to do this o Human genome codes for hundreds of different ABC transporters o Two domains

Transmembrane domain show in teal ATP binding cassette shown in purple

Different transporters transport different things

ATP binding domain simply binds ATP and uses the energy of ATP breakdown to fuel the transport

o Specificity is in the transmembrane domain, not the ATP binding domain 13. Role of electrochemical gradient in determining equilibrium concentration of ions

There are two types of gradients working in a cell, a chemical gradient and an electrical gradient

The chemical gradient is caused by different levels of concentration of certain molecules on either side of the membrane

The electric gradient is caused by different charges on either side of the membrane

Molecules want to flow to the lower concentration but they are pulled back by their charges

Equilibrium is reached where there is a happy medium between both gradients

The abundance doesn’t necessarily have to be the same on both sides 14. Basis for electrical gradient across photoreceptor cell

Plasma membrane of the photoreceptor cell in the dark

There is a charge difference across the membrane o There is lots of anions inside the cell

Amino acids, proteins o Lots of sodium outside the cell and potassium inside

They leak in/out of the cell down an electrochemical gradient

Facilitated diffusion They are pumped through a sodium-potassium active transport complex back to

where they came from

cGMP-gated channel o facilitated diffusion o When it binds, you get a huge influx of sodium which keeps the difference across the

membrane small than it would otherwise be: -30mV

In the light, cGMP is metabolized (shut off) by the phosphodiesterase

The positively charged sodium can’t get into the cell and minimize the negative charge, so it becomes hyperpolarized: -60 mV

o That’s the trigger that fires down the rest of the cell, blocks the release of Glutamate and triggers the electrical impulse

15. Basic structure of ABC transporter o Two domains

Transmembrane domain show in teal ATP binding cassette shown in purple

Different transporters transport different things

ATP binding domain simply binds ATP and uses the energy of ATP breakdown to fuel the transport

o Specificity is in the transmembrane domain, not the ATP binding domain 16. Genetics underlying cystic fibrosis

Homozygous recessive disease caused by a mutation to CFTR (ABC transporter)

CFTR is a gigantic protein – 6000 bases, 1480 amino acids

17. Cystic fibrosis phenotype

Homozygous recessive

Most common mutation is ΔF508 (70% of cases)

o Deleted a phenylalanine at position 508 – type of amino acid

18. Physiological function of CFTR and sodium transporter

People with CF suffer from problems from their lungs and gastrointestinal tract

Mucus and cilia must be kept wet so you can cough out bacteria and dirt o Kept wet by the action of CFTR which pumps Cl- ions out into the epithelial and in

response to that you get osmotic movement of water Water moves from epithelial cell to epithelial lining

o Otherwise you have lung infections, gas exchange is inhibited (diffusion of oxygen is difficult)

19. Relationship of CFTR synthesis and folding in the intra-cellular secretory system

CFTR is missing a piece in the mutant form which causes it to fold improperly - ΔF508

To make it to the membrane it must pass from the ribosome, through the ER and the golgi

In the ER there is a quality control mechanism that sees if everything is folded properly 20. What happens to the deltaF508 form of CFTR

It gets degraded in the ER and the proteasome (full of proteases which break down proteins) breaks down the protein

The ΔF508 form is perfectly functional! o Not as good as the wild type form o Can synthesize it, it still pumps chlorine

About 50% as well as the wild type o If you could get the defective protein to the plasma membrane then you wouldn’t have

CF It doesn’t get there because it gets tagged in the ER

21. Role of chaperone proteins

Detect fault in proteins

Proteins that assist the non-covalent folding or unfolding and the assembly or disassembly of other macromolecular structures

Do not occur in these structures when the structures are performing their normal biological functions having complete the processes of folding and/or assembly

22. Role of proteasomes

Degrade proteins that do not have the correct shape by proteolysis, a chemical reaction that breaks peptide bonds

Enzymes that carry out such reactions are called proteases

Lecture 6 Outcomes

1. Definition of:

Photosynthesis o The process by which organisms use light to synthesize foods from carbon-dioxide and

water

Oxidation o A chemical reaction in which there is the loss of electrons or gain of oxygen, hence

resulting in an increase in oxidation state by a molecule (oxidation of glucose produces CO2)

Reduction o Electrons are added to an atom or ion (by removing oxygen or adding hydrogen)

always occurs with the oxidation of the reducing agent

Oxidation-reduction reaction o An electron is transferred from one molecule to another o The electron-donating molecule is the reducing agent or reductant; the electron

accepting molecule is the oxidizing agent or oxidant

Light reactions o Biochemical reactions in photosynthesis that require the light energy that is captured

by light-absorbing pigments (chlorophyll) to be converted into chemical energy in the form of ATP and NADPH

Calvin cycle o A series of biochemical reactions that occur in the stroma of chloroplasts during

photosynthesis o It includes the light-independent reactions such as carbon fixation, reduction

reactions and RuDP whereby sugars and starch are ultimately produced

Redox potential o The reducing/oxidizing power of a system measured by the potential at a hydrogen

electrode o The ability of an electro to act as a reducing or oxidizing agent via its positive or

negative charge

Chloroplast o The chlorophyll-containing plastid found within the cells of plants and other

photosynthetic eukaryotes

Thylakoid membrane o Part of the chloroplast, found as part of the grana and also as single cisternae

interconnecting the grana o Contains the photosynthetic pigments, reactions centers and ETC

Lumen o Inside space of a tubular structure, continually aqueous plays a vital role

photophosphorylation o Protons are pumped into the lumen making it acidic

P680 o The reaction centre of photosystem II, primary donor

P700 o Reaction centre of photosystem I

(+ and * for each) o * means excited state of the photosystem, where the redox potential shifts to more

negative o + means that it has been oxidized and now has the ability to rip an electron away

Chemiosmosis o The diffusion of ions across a selectively-permeable membrane

Phases of Calvin cycle o Phase 1 – carbon fixation

CO2 is incorporated into a 5 carbon sugar named RuBP, the enzymes that catalyzes is rubisco

The product is 3PG

o Phase 2 – reduction ATP and NADPH2 from light reaction are used to convert 3PG to G3P

o Phase 3 – regeneration More ATP is used to convert some of the G3P back to RuBP, the

acceptor for CO2 2. Structure of chloroplast

Surrounded by a double membrane

The stroma is filled with aqueous compartments – where Calvin cycle takes place

Thylakoid stacks are called granum

The thylakoid stacks have chlorophyll molecules on the surface (photosystems) where light reactions take place

Inside the thylakoid is the thylakoid lumen – totally membrane enclosed 3. Source of electrons and products of electron transport

P680 is oxidized and gets its electrons from the splitting of water, happens in the lumen side of the oxygen involving complex

The products are ATP, NADPH, and oxygen 4. Structure of photosynthetic electron transport

Large supra complexes found in the thylakoid membrane

There is Photosystem 1 (P700) and Photosystem 2 (P680) o Both have reaction centers surrounded by an antennae which has light absorbing

molecules

In between the photosystems is the cytochrome complex and there is ATP synthase

PQ is an electron transporter that shuttles electrons

5. Mechanism of electron flow

Photosystem 2 on the left, photosystem 1 on the right and cytochrome complex in the

center

Both PS2 and PS1 have a reaction center that is surrounded by an antenna o Light gets funnelled to the reaction centre o Reaction center chlorophyll of photosystem 2 (P680) and photosystem 1 (P700)

The reaction center chlorophyll gets oxidized and the electron leaves and travels down an electron transport train to photosystem 1 o PQ takes the electron and becomes negatively charged, and also picks up a proton

from the stroma so it becomes neutral and can move through the cytochrome complex

o Dumps the proton in the lumen creating an electrochemical gradient Can use the gradient to do work The protons get back through ATP synthase harnessing the energy from

the electrochemical gradient

Generates ATP

Maintaining membrane fluidity is important – PQ could fall apart if too fluid, could have trouble moving rapidly if too rigid

Electrons move because of the redox potential difference between electron carriers

For ETC to work, it must accept an electron then hand it off

If you have a move positive redox potential, you are a very strong oxidizing molecule o You have the ability to pull electrons away from other molecules

If you are more negative, you don’t hold onto electrons very strongly and are readily able to reduce other molecules

This flow is spontaneous – based on redox potential

Light in photosynthesis changes the redox potential of the reaction centre chlorophylls o Makes the redox potential more negative

P680 absorbs a photon of light and now in the excited state it is easy for it to give the electron away – changed the redox potential by absorbing light

Need 2 photons to get all the way to the negative redox potential of NADP in photosystem 1

6. Phases of Calvin cycle

Occurs in the stroma

3 turns of the cycle – it fixes 1 CO2 per turn

CO2 Fixation o Carbon in CO2 is incorporated into RuBP (5 carbon sugar) o The enzyme that catalyzes is rubisco o The product is 2PG

Reduction o Requires reducing power so NADPH is required to reduce the carbon o 3PG is converted

to G3P

Regeneration of RuBP o More ATP is used

to convert some of the pool of G3P back to RuBP, the acceptor for CO2

7. Major carbon compounds of Calvin Cycle - Ribulose bisphosphate (RuBP), phosphoglycerate (PGA), glyceraldehyde 3-phosphate (G3P)

RuBP is a 5-carbon chemical that combines with CO2 at the beginning of the Calvin Cycle

PGA is formed when the CO2 is fixed to RuBP

G3P is formed from the reduction of PGA with the power of ATP and NADPH 8. Stoichiometry of carbon in Calvin Cycle (major molecules listed above)

RuBP is a 5 carbon sugar (15 carbons), it binds with CO2 (3 carbons) to make the 6 carbon sugar citrate (18 carbons)

It produces two, 3 carbon molecules of PGA (18 carbons)

PGA is reduced to G3P (18 carbons)

G3P is regenerated into RuBP which is a 5 carbon molecule (15 carbons) 9. Reaction catalyzed by rubisco

Carbon fixation is where CO2 is added to a molecule of RuBP to make a 6 carbon molecule called citrate o The carbon is conserved in the citrate molecule and is used to make two 3-carbon

molecules of PGA 10. Difference between carboxylation reaction and oxygenation (photorespiratory) reaction

Every carbon in us at one time passed through the active site of rubisco at one time or

another

Carbon gain = growth

o You’ve gained weight so that you can grow

Oxygen can compete with carbon dioxide for the active site of rubisco

o Competitive inhibition

o When oxygen gets into the active site it catalyzes photorespiration (oxygenation of

rubisco)

o There was no oxygen around when rubisco developed

The selective pressure to design an active site that wouldn’t allow

oxygen to bind there wasn’t present because oxygen wasn’t around

then

o If oxygen reacts with RuBP you don’t get more carbon so you don’t grow

11. Implication of photorespiration of growth

Photorespiration happens when there is a lack of CO2 so there is no carbon fixation and the plant cannot grow

12. Mechanism by which Chlamydomonas concentrates carbon dioxide

Aquatic photoautotrophs o HCO3

- is the form CO2 is in in an aqueous environment o There is a pump that pumps in bicarbonate so levels of

it are very high in the cell o In there you have the enzyme that has the greatest

catalytic turnover rate know – carbonic anhydrase o Converts bicarbonate into CO2 which diffuses down a

concentration gradient into the chloroplast

This is how they minimize photorespiration

Lecture 7 Outcomes

1. Characteristics of ATP

ATP is a nucleotide

One unit made up of on adenosine molecule with three phosphate connected to it

Forms ADP in water and the formation releases energy in a form known as hydrolysis

ATP also has a role in transferring energy within the cell, moving the energy from chemical bonds to actual energy reactions

2. Role of C-H bond in bioenergetics

There are a lot of C-H bonds in the food we consume (carbs, fats, proteins)

There is a lot of free energy in C-H bonds that can be converted into energy that is useful for the cell to use

Energy can be conserved from the electrons in the C-H bonds 3. Role of redox potential in bioenergetics

Negative redox potential – gives up electrons (readily oxidized)

Positive redox potential – attract electrons (readily reduced)

Oxygen has a very positive redox potential and wants the electrons

The electrons move in the ETC because the complexes in the membrane are organized by redox potential o Flows from negative to positive o Oxygen is the terminal electron acceptor

4. Role of FAD, NAD+ as electron carriers

Both molecules readily accept an electron pair and they can also readily donate an electron pair

When electrons are transferred, molecules of hydrogen are also transferred (a proton)

This happens in the breakdown of Acetyl CoA in the Krebs Cycle (aka the citric acid cycle) inside the second membrane of the mitochondria

NADH and FADH2 will carry and donate the electrons to the electron transport chain on the internal membrane

The transfer of the electrons energy allows for the proteins crossing the membrane to pump hydrogen ions into the space between the two membranes and build up a gradient for chemiosmosis

The NADH and FADH2 become NAD and FAD again and return to the Krebs Cycle 5. Location, products, distribution in nature and purpose of pathways such as glycolysis, CA

cycle, respiratory electron transport etc.

Glycolysis o Location – cytosol o Purpose – take glucose and turn it into two molecules of

pyruvate that can enter the matrix o Products – 2 ATP and 2 molecules of pyruvate

NADH for every glucose molecule o Exergonic reaction (sponteanous)

Pyruvate Oxidation o Location – mitochondrial matrix o Pyruvate loses a carboxyl group (CO2), forms NADH and through the addition of

coenzyme A because acetyl CoA (2 carbons) Since 2 pyruvates are turning into

2 acetyl CoA (4 carbons)

Citric Acid Cycle o Location – matrix of the mitochondrial cell o Products – FADH2, NADH, ATP and G3P o Purpose – to create NADH and FADH2 to help

with electron transport and OXPHOS o For the exam – what does it do, where is it

found?

Oxidative Phosphorylation o Location – occurs ON the inner mitochondrial

membrane o Products – ATP, NAD+/FAD+ o Purpose – to use electron

transport through the different redox potentials and to use the electrochemical gradient to pump protons through the membrane and create ATP

6. Role of energy coupling in early steps of glycolysis

When glucose and hexokinase come together with a phosphate group you get G6P o Endergonic reaction (nonspontaneous)

If you couple it with an exergonic reaction, the exergonic reaction will overpower the reaction

The exergonic reaction is the breakdown of ATP binding to the active site in hexokinase

Very first reaction in glycolysis

Hexokinase o Adds a phosphate to position 6 of the glucose o Not a spontaneous reaction – cannot occur by itself

Half reactions

o Pi + glucose glucose-6-P

ΔG = +3.3 kJ/mol

o ATP + H2O ADP + Pi

Δ= -7.3 kcal/mol

Coupled reaction:

o ATP + glucose ADP + glucose-6-P

ΔG = -4kcal/mol

o The coupling of the reactions gives it an overall – ΔG so the reaction occurs

Thermodynamically it is unstable, but kinetically it is stable

o ATP doesn’t hydrolyse by itself

No real hydrolysis is going on, better to call it ATP breakdown

o The free energy of the phosphate is transferred to glucose

But why would you want to phosphorylate glucose?

o A phosphate group is charged, glucose isn’t so it can travel through membranes

Adding a phosphate doesn’t allow it to pass through membranes

o Makes the glucose more reactive – the negative charge makes the molecule more

unstable

7. Relative potential energy of various intermediate compounds (eg. glucose vs. pyruvate vs. CO2)

Glucose has a higher potential energy than pyruvate because it has been oxidized o The potential energy can be used to produce ATP o It can be stored for later as glycogen

The potential energy of pyruvate is lower than glucose but is used to drive the Citric Acid Cycle

CO2 doesn’t have any energy and needs to be oxidized to give energy to molecules 8. Reasons why catabolic intermediates are phosphorylated

o Catabolic intermediates such as glucose are phosphorylated because it makes them more reactive – more readily wanting to break down

Glucose-6-phosphate is more reactive and has more free energy associated with it than glucose does

o Gives the molecule a negative charge so it cannot diffuse through the membrane 9. Link between glycolysis and Citric Acid Cycle

Occurs in the mitochondrial matrix

Pyruvate has C-H bonds so it makes sense that you want to oxidize it further

No free energy in the carboxyl group is it decarboxylates it – decarboxylation o Loses the CO2

Dehydrogenase catalyses the NAD+ to NADH

Adding coenzyme A will make the molecule more reactive

This creates Acetyl CoA 10. Role of pyruvate dehydrogenase complex

PDC is a big enzyme complex that transforms pyruvate into Acetyl CoA

The remaining energy in Acetyl CoA is extracted by the Citric Acid Cycle 11. Diagnostic value of relative ratios of bioenergetic intermediates (eg. ATP, pyruvate, NAD etc.) 12. Relative location of electron transport chain components relative to mitochondrial

membrane, matrix, intermembrane space

The components of the electron transport chain are located on the inner mitochondrial membrane

A majority of the reactions take place in the mitochondrial matrix and the proton by-products are shuttled to the intermembrane space

13. Role of oxygen in electron transport

Process of redox reactions occur as electrons are passed along a chain of electron carriers until they are finally passed to oxygen o The final electron acceptor

Oxygen has the most positive redox potential and thus can be easily reduced

When electrons reach oxygen, oxygen is reduced by two electrons and turns into water o Oxygen is responsible for removing electrons from the system o if there was no oxygen, electrons could not be passed among the cofactors and the

energy in the electrons could not be released to do work oxygen is necessary to “drain” electrons from the system

14. Role of NADH in electron transport

NADH freely donates electrons and oxygen is the terminal electron acceptor o Lots of free energy in NADH o This free energy is used to do works

It donates electrons to the mobile carrier UQ 15. Role of cofactors in ETC

Cofactors are bound to protein subunits in complexes I, III, and IV

Cofactors are redox-active cofactors that alternate between reduced and oxidative states as they accept electrons from upstream molecules and then donate electrons downstream to molecules

Cofactors essentially carry the electron donated from NADH all the way to oxygen

Each successive cofactor has a more positive redox potential so that it can oxidize the cofactor before it

16. Relationship between redox potential of ETC intermediates and “flow” of electrons

They move from negative to more positive redox potential

Move down the chain to oxygen which has a very positive redox potential

This movement is thus spontaneous 17. Link between ETC and synthesis of ATP

The energy of the electrons released during ETC is used to do work

The work of transporting protons across the inner mitochondrial membrane from the matrix to the inter-membrane space

The electrochemical gradient formed can be used to do work (chemiosmosis)

ATP synthase then catalyzes ATP synthesis sing energy from the H+ gradient across the membrane

18. Effect of uncoupling agents on ATP synthesis

Uncoupling agents inhibit the production of ATP

Uncouplers give protons an alternate route of getting back to the mitochondrial matrix

A proton is much more likely to enter through the uncoupler than through ATP synthase, and without protons entering ATP synthase, no ATP is synthesized

Uncouplers can allow for high rates of electro transport but prevent chemiosmotic ATP synthesis

If this happens, the energy released during electron transport is not used to do work, it is released as heat instead

Newborn kids and hibernating animals can use uncouplers to generate heat and regulate body temperature (less energy, more heat)

If there is low expression of uncouplers, metabolic rate would go up and this accounts for a higher rate of oxygen consumption o Oxygen is continually reduced and turned into water and no ATP synthesis is made

Leads to obesity (body continually needs more energy and so it eats more – eats lots of sugars that supply quick energy)

19. Goal of making lactate under conditions of hypoxia

Hypoxia = deficient oxygen

When oxygen is absent or in short supply, the pyruvate molecule does not enter the mitochondrion (stays in the cytosol) and as a result, NADH is not oxidized (no NAD is regenerated)

With no oxygen, NADH cannot be oxidized and ETC will stop

In lactate fermentation, pyruvate (3 carbons) is converted into the 3 carbon compound lactate

This conversion takes in NADH (and one proton) but replenished NAD+ so that glycolysis can occur under low oxygen

20. Role of NAD+/NADH in sensing hypoxia

Redox homeostasis o NAD+/NADH ratio o This ratio tells a lot about what’s going on with oxygen in our cells o If you have lots of NADH around, glycolysis and citric acid cycle are working, but

suggests an inhibition of electron transport because one of the outcomes of electron transport is to oxidize NADH

o Insufficient oxygen would lead to this ratio being very low (lots of NADH) 21. Role of HIF1 regulation in sensing hypoxia

HIF-1alpha is synthesized and located in the cytosol

If there is a lot of oxygen in the cell, HIF-1alpha never makes it to the nucleus, it gets degraded

It gets hydroxylated and it tagged by a molecule called ubiquitin and is sent to the proteasome to be degraded

Under low levels of oxygen, it is NOT degraded, so it is migrated to the nucleus, binds to the HIF-1beta transcription factor where it makes a function dimer called HIF-1 transcription factor (it’s a protein)

22. Effects of HIFI activity on pyruvate metabolism

HIF-1 transcription factor activates a gene which codes for pyruvate dehydrogenase kinase – this kinase is synthesized, and then goes to block the pyruvate dehydrogenase complex (the cluster of enzymes in the mitochondria)

This inhibits the metabolism of pyruvate and so pyruvate does not enter the mitochondria – lactate fermentation occurs instead

23. Feedback regulation of glycolysis, CA cycle and ETS (not covered in class)

Glycolysis o Availability of substrate o Concentration of enzymes responsible for rate – limiting steps o Allosteric regulation of enzymes o Covalent modification of enzymes (ex. Phosphorylation)

CA Cycle: o Product inhibition and substrate availability

HAND inhibits pyruvate dehydrogenase Calcium activates pyruvate dehydrogenase phosphate which in turn

regulated the complex

Increases flux throughout the pathway o Citrate is a feedback regulator that inhibits an enzyme in glycolysis to minimize the

amount of pyruvate made

ETC o Uncouplers and couplers o The amount of NAD+/NADH o Amount of O2

24. Various characteristics that compare/contrast with those of photosynthesis

Similarities o Both involve electron transport chains o Chemiosmosis allows ATP synthase to produce ATP o Both utilize ATP for energy at some points o Both provide power for cellular activities

Differences o Cellular respiration depends on oxygen as a substrate o Photosynthesis utilizes 2 electron transport chains (not just 1) o In photosynthesis, energy is provided by photons and not catabolic processes as in

cellular respiration o Photosynthesis involves the production of NADPH – cellular respiration involves NADH

and FADH2 o Photosynthesis involves CO2 and H2O as substrates (splitting H2O provides electrons

for the process)

Lecture 8 Outcomes

1. Change in respiration rate (oxygen consumption) in isolated mitochondria by addition of NADH, ADP, uncoupler etc.

Mitochondria – added at 2 minutes o If mitochondria is added to an oxygen electrode chamber, the oxygen is

consumed so there is a slight drop in the line

NADH – added at 4 minutes o Oxygen consumption increases even further o Leads to more electron transport and a greater need for oxygen

ADP – added at 6 minutes o Even greater increase in oxygen consumption o Without ADP, ATP cannot be synthesized and thus proton gradient becomes

VERY high and the pH in the inner membrane compartment becomes very low o It becomes harder to pump protons into a space that already has a lot of

protons and this limits the rate of ET o Once ADP is added ATP can be synthesized and ET can resume to an enhanced

efficiency. This is known as respiratory control

Uncoupler – added at 8 minutes o Completely gets rid of the concentration gradient o Without the concentration gradient, protons are pumped much quicker and

electron transport as a whole operates much faster o This leads to an increase in oxygen consumption (but NO ATP IS MADE)

2. Definition of respiratory control and how it is accomplished (proton gradient)

Respiratory control – rate of electron transport will be limited or dependent upon the availability of ADP (substrate)

When there is no ADP, ATP cannot be synthesized, as a result the concentration gradient continues to build making it harder for protons to be pumped into the inner-membrane compartment which slows electron transport, inhibits ATP synthesis and slows down oxygen consumption

o This ensures substrates will not be oxidized wastefully

When ADP is added, oxygen uptake proceeds at an enhanced rate until all of the ADP added has been converted to ATP

3. Metabolic link(s) between chloroplast and mitochondria

The metabolic link between chloroplast and mitochondria in Chlamy cells is REDUCED CARBON

o Light reactions use ADP to make ATP…. ATP is fed into the Calvin Cycle and comes out as ADP (which is then used in light reactions)

So basically, ATP stays within the chloroplast and is not exported

How does the mitochondria get carbon to make ATP in Chlamy if it doesn’t eat? o It gets reduced carbon from the Calvin cycle

Calvin Cycle makes G3P which exists through a transporter on the chloroplast into the cytosol

4. Reasons why Chlamydomonas can grow as a heterotroph on certain reduced carbon compounds - but not others.

Chlamy can’t grow as a heterotroph on glucose because it does not have a glucose transporter – it cannot pass Chlamy’s membrane

HOWEVER, Chlamy can grow heterotrophically (it can live in the dark) o The molecule it can grow on in the dark is acetate (it has 3 C-H bonds which it

can use for energy) o There is an acetate transporter on Chlamy’s membrane that can bring it in o Once in the cytosol it can be converted to Acetyl-CoA

Lecture 9 Outcomes

1. How to measure carbon fixation in Chlamydomonas

Put Chlamy cells in a CO2 analyzer 2. How one can distinguish between gas exchange in mitochondria from that taking place in the

chloroplast of a Chlamydomonas cell

Measure the CO2 fixation rate of Chlamy when the lights are off o In the dark there is no photosynthesis occurring

We can assume that this dark rate is constant – mitochondrial gas exchange = dark level 3. Identify major parts of a light response curve for carbon fixation

The CO2 fixation rate will increase linearly as the light intensity increases

The rate is linear because the rate of the Calvin Cycle is directly proportional to the products of the light reactions

4. What metabolic processes and external factors may influence the change in rate as a function of light

The linear portion of the curve is limited by NADPH and ATP supplies (products of the light reactions) – needed to work the Calvin Cycle

As light intensity increases, the following limit the rate of carbon fixation o The speed at which the enzymes in the Calvin Cycle can work

The speed of Rubisco o Turnover rate of the enzymes

The speed at which RuBP can be regenerated o If not enough CO2 is available, the speed at which CO2 can be fixed is hindered

o Once the curve begins to plateau, any light supplied past this point is considered excess light that can be damaging – proteins that P680 and P700 are attached to could break down

5. Light compensation point

The point at which the rate of CO2 being brought in by the chloroplast matches perfectly with the amount of CO2 being released by the mitochondria is known as the light compensation point )

o When CO2 fixation rate = 0

In order to have a healthy plant, you must be above the light compensation point o Need to have a net gain in carbon for growth

6. Principal of measuring enzyme kinetics as a function of substrate concentration

To measure the rate at which enzymes make product, the enzyme concentration MUST be kept constant

You can have different tubes with different substrate concentrations and see how fast the enzymes work

o This can be observed through colour

As you add more substrate, the enzyme can process more and thus the velocity of the reaction goes up

At a certain amount of substrate added, the velocity plateaus because the enzyme has reached its maximum speed

7. km, Vmax

The measure of the interaction between the substrate and the enzyme is called Km

Km is the substrate concentration that gets 1/2Vmax

Km is a measure of affinity – the attractiveness between substrate and the enzyme complex

If you have high Km, you have LOW affinity – you need more substrate to reach the same speed that an enzyme would reach with less substrate

If you are efficient at getting substrate and binding it, you have a very low Km

The grater the Km the less affinity between the enzyme and the substrate 8. Effect of a competitive inhibitor on enzyme kinetics (Vmax, Km)

When an inhibitor is added, the Vmax does not change o There is so much substrate that an inhibitor is irrelevant

BUT, when an inhibitor is added, the Km becomes larger o If 3 inhibitors are added to 4 molecules of substrate this will affect the ½ Vmax

due to the high ratio) As a result the Km shifts right

At high levels of substrate (20 molecules of a substrate and 3 inhibitors) Vmax stays the same because there is so little inhibitor compared to substrate that there is no difference

So Vmax and ½ Vmax is the same for normal catalysis and for catalysis with an inhibitor

However, Km is greater when there is an inhibitor (in respectable amounts) o A competitive inhibitor competes with a substrate for an enzyme’s active site, it

lowers the enzyme’s likelihood of binding substrate (less affinity) and slows the reaction velocity, but leaves the actual amount of end product unchanged

9. Structure of peptidoglycan

Bacteria cell wall is made out of peptidoglycan – a combination of glycan (complex carbs – the blue and purple hexagons) and peptide chains that link the whole system together

When bacteria replicates and needs a new cell wall, transpeptidase is the enzyme that fuses this cell wall together

o Without transpeptidase you cannot make peptidoglycan

Transpeptidase is a bacterial enzyme that brings two amino acids groups (carbs) at the ends of two peptides together and links them up

10. How penicilin mimics structure of peptidoglycan to inhibit transpeptidase function

Penicillin looks identical to the two amino acids used by transpeptidase

Penicillin tricks transpeptidase and gets into its active site and competitively inhibits the enzyme

That’s why when you’re on penicillin you need LOTS of it so that it can outcompete the regular substrates and so it binds to transpeptidase and ‘destroys’ it