1011 FINAL EXAM SET W5-W12

What are proteins

• Proteins are complex molecules

• Carry out almost all biological functions

Figure out the nature of Inhibitors 1 and 2

Inhibitor 1 is uncompetitive → we can see this because it is parallel to the enzyme line (no inhibitor)

Inhibitor 2 is competitive → we can see that is will intercept the enzyme line (no inhibitor) line at the same y-axis, this means the Vmax is the same at both conc of substrate, which resemble the graph for competitive inhibition

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CALCULATE Km and Vmax for Inhibitor 1

Y-intp → 0.04 therefore the Vmax is 1/Vmax = 1/0.04 = 25.00

X-intp → -0.167 therefore the Km is  -1/Km = -(1/-0.167) = 6.00 mM

*When finding the x-intp, make sure you set y=0 and solve for x - BE CAREFUL

Calculate the Km for Series 1

1. Infer the Vmax to be about 30μmol/min (this is where the graph should start to plateau out)

2. Half of Vmax is 15μmol/min

3. Trace the graph to see the SUBSTRATE CONC at 15μmol/min

4. This will give about 6mM

How is such diversity of protein functionality achieved?

Protein FUNCTION is dependent upon STRUCTURE, which is dependent upon S-E-Q-U-E-N-C-E

What is the hierarchy of structure in proteins?

• Rotation around the peptide bonds in the backbone of a protein means countless possible conformations are possible → functional conformation are termed ‘native’

  • These ‘conformations’ are when the protein can perform a function

• Early observation showed that proteins could be crystalized this suggested that protein had similar 3D structures

What are amino acids?

• Monomers that make up proteins

• The backbone of AA is the N, the alpha C and the C in the carbonyl (3 atoms)

• There are 20 common AA

• Key function of AA:

  • Capacity of polymerise

  • Useful acid-base properties

  • Varied chemical and physical properties (physicochemical)

• Hydrophobic amino acids are largely buried in the protein interior so that the number of hydrogen bonds and ionic interactions within a protein is maximised

• Leucine, Valine and Isoleucine are considered BRANCHED amino acids (BCAAs) - they are essential amino acids, the body cannot synthesise these and they must be obtained from the diet

Peptides

• Due to electron RESSONANCE the C-N bond has a double bond like characteristic

• This makes the bond quite rigid and the CO and NH apart of the peptide become quite planar - no rotation in the peptide bond

• We only see rotation on the alpha carbons near the peptide bond (in blue) (hence the R groups orientation)

• If asked for the number of amino acids in a polypeptide - COUNT THE R-GROUPS or count the peptide bonds and ADD 1

How does structure arise?

• Largely by weak interactions (REVERSIBLE)

  • Hydrogen bonds

  • Ionic interactions

  • Hydrophobic effects

    • The hydrophobic effect predominates in protein structure and stability

  • Van der Walls

  • The release of water molecules from the structured solvation layer around the molecule, as the protein folds, increases the net entropy

• Covalent bonds in the peptide backbone also limit structural conformations

Importance of Hydrogen Bonds

• H-bonds are dipole-dipole or charge-dipole interactions

  • Linear H-bonds are stronger than bent ones

• They arise between a covalently bound hydrogen (H-bond doner) and lone pairs of electrons (H-bond acceptor)

• They typically involve two electronegative atoms (frequently nitrogen and oxygen).

What is the arrangement of amino acids in the Secondary structure?

• Several common and stable structure types are observed across proteins (and different amino acids favour different structures)

  • The Alpha Helix - the R groups determine surface chemistry

  • The Beta conformation - SHEET

  • The Beta turn

Secondary structure: Alpha Helix

• Backbone atoms wind in right-handed helix

• Backbone is twisted (not extended)

• One turn every 3.6 residues

• Each residue ~100 deg rotation

• R groups are all on the outside (B) - helps with stablisation (e.g. ionic bonds that could form (D))

• Intrachain H-bonding most important stabilizing feature

• CO of res i is H-bonded to NH of res i+4

  • 1 will go with 5

  • 2 will go with 6

Secondary Structure: Beta Sheets

• Extended and more of a zigzag conformation than a helix

• Many beta-strands comes together to form a b-sheet

• Stabilized by H-bonds

• Arrow head is always the C-terminus

• Note that the R groups of adjacent amino acids protrude in opposite directions and are above and below the sheet

  • If the R groups point the same way → parallel, straight H-bonds

  • Opposite ways → anti-parallel, BENT H-bonds, hence STRONGER

Secondary Structure: Beta Turns

• The U-turn of proteins

• Allows 180° turn

• Connects two ends of anti-parallel beta sheet

• Involves four amino acids (specific residues vary)

  • Glycine is very common at position 3 (type II)

  • Proline common at position 2 (type I)

    • Proline has a forced kink that helps with forming Beta-turns

How to display the 3D model of protein structure?

• Notice how the hydrophobic parts are often in the interior of the protein (in order to maximize the strong H-bonds)

The disulphide bond

• Covalent cross-link (bridge) by two amino acid cysteine to form a cystine

• Can be intrachain or interchain

• Is reversible (depending on the oxidizing or reducing environment)

What are the major classes of tertiary structure?

Fibrous — Simple, (usually) repeating secondary structure

• Often function in structure for support, shape, protection

• Hydrophobic

• Keratin, Collagen

Globular — Several types of secondary structure, more folded, more compact

• Most other stuff…regulatory proteins, enzymes

• Myoglobin, Haemoglobin

What is Quaternary structure?

• A protein with two or more separate polypeptide chains (subunits) - includes 2 (dimer), if 3 it would be a trimer

• Subunits can be identical (homodimer) or different (heterodimer)

What are protein motifs?

• They are a recognisable folding pattern, linking two or more secondary structures together

• Also called a fold or supersecondary structure

What are protein domains?

• These are independently stable parts of a polypeptide chain, or those that can undergo movement as a single entity with respect to the protein

• For example the calcium binding domain of this protein

• E.g. 1 domain → active site, 2nd domain → the active/inactive binding site (imagine tryptophan regulator protein)

What happens when proteins don’t fold correctly?

• Loss of function

• Aggregation (Parkinson’s, Alzheimer’s)

• These are all in equilibrium systems

• Autophagy is when the protein eats itself to try and stop this aggregation

What are the requirements for a protein to fold into its native conformation?

Experiments conducted by Christian B. Anfinsen (1916-1995)

• He used RNAase A and questioned:

  • Is the native conformation of RNAase A required for it to be functional?

  • What are the requirements for RNAase A to fold into its native conformation?

• NOTE the correct disulphide bonds are required

What factors influence the NATIVE formation of the native conformation of proteins?

• Disulphide bonds (covalent, so strong and stable)

• Weak interactions:

  • Non-covalent; weak, but overall very stabilising (reversible → breaks and forms over and over again until he most stable form is formed)

  • Hydrogen bonds, electrostatic interactions, Van der Walls

  • Hydrophobic effect – burying of hydrophobic groups

• Enthalpy

  • Formation of bonds/interactions decreases enthalpy of a system (favorable; happens spontaneously)

  • As bonds are formed they release energy into order to stablise the protein → decreases enthalpy

• Entropy

  • Protein structure is a more ordered state (low entropy), so entropy opposes formation of interactions (but … it’s all about overall change in free energy) INCREASE

Protein fold via stepwise pathways?

• Secondary structure elements form first

• These then fold against each other (various intermediates along the way)

Protein folding and Entropy

• A long, floppy unfolded protein has lots of possible shapes → high entropy

• When it folds into one specific shape, it becomes more ordered → entropy decreases

• Nature hates losing entropy.

  So:

  • Folding decreases entropy = bad for folding

  This is why entropy “opposes” folding

• When hydrophobic amino acids get buried inside the protein, water molecules are freed from their ordered cages around them.

• This increases the entropy of water, which is a huge driving force for folding

• So even though the protein becomes more ordered, the water becomes less ordered, and that helps folding happen.

Examples of Fibrous proteins

Membrane proteins

• Look at how when you approach the hydrophobic fatty acid tails, the amino acids are hydrophobic in the middle, and then on the top and the bottom we have the polar amino acids again

Globular protein - Myoglobin

• Found mainly in muscle tissue, where its role is to bind to oxygen (delivered to tissue via hemoglobin in the blood) and supply it to mitochondria

• It's quite a small molecule. It only has a single polypeptide chain of 153 amino acids. TERTIARY STRUCTURE

• The structure has 8 α-helices, along with a pocket that binds porphyrin ring (shown in red) that has an iron bound at its centre

• The iron is what binds oxygen

• This iron-bound porphyrin ring is known as "heme" and is technically a "prosthetic group"(A prosthetic group is a compound permanently associated with a protein that helps with the protein's function.)

  • It's actually the side chain nitrogen of a histidine (His) amino acid that helps to interact with the heme:

• Myoglobin binds oxygen with high affinity, and this property helps it to acquire the oxygen from incoming molecules of haemoglobin

Globular protein - Haemoglobin

• QUANTERNARY STRUCTURE

• four polypeptide chains (two alpha chains, two beta chains). Each is very similar in sequence and structure to myoglobin.

• picks up oxygen from the lungs and transports it to tissues, where it hands it off to myoglobin

• Haemoglobin's structure is critical to its function. Binding of oxygen to one subunit increases its affinity for oxygen in the other subunits. This occurs due to structural changes in the molecule.

  • Without oxygen, haemoglobin is said to be in the "T state" (think "tense"; it's really tense because it's desperate to have oxygen)

  • When oxygen binds, there is a subtle shift in the conformation of the surrounding amino acids, and it shifts to the "R state" (think "relaxed"; it can breathe a sigh of relief now it has oxygen!)

  • This is a really subtle change, but it has a knock-on effect to the adjacent subunits, making them more amenable to oxygen binding

  • This is known as cooperativity, or cooperative binding. Binding of a ligand (in this case oxygen) to one subunit impacts the ability of another subunit to bind. In this case, the impact is positive

  • What's the point of all this? It means that haemoglobin is more likely to bind oxygen when it is in plentiful supply (in the lungs) but will release that oxygen when it is in an environment of lower oxygen concentration (the tissue). It allows a trade-off in affinity for oxygen, aiding its function.

The Graph

• The "High-affinity state" line depicts the situation if haemoglobin permanently adopted a high affinity (i.e. a high capacity to bind) oxygen. Even in the low-oxygen environment of tissues it would still be holding on tight to oxygen, preventing it from effectively reaching the cells of the tissue.

• The "Low-affinity state" line depicts a situation if haemoglobin always had a low affinity (i.e. a low capacity to bind) for oxygen. If this were the case, it would readily let go of oxygen in tissue (desirable), but wouldn't be able to acquire much oxygen in the lungs (undesirable)

• The reality is that due to cooperativity of binding, haemoglobin can shift between high-affinity (in the lungs) to low-affinity (in the tissue), all due to the amount of oxygen in the environment. This is shown in the middle line labelled "Transition from low- to high-affinity state".

What are enzymes and why do we need them?

• Enzymes increase the rate of (catalyse) biochemical reactions - they are biological catalysts

• They are essential to life - reactions would occur too slowly without enzymes

• Many enzymes are part of coordinated pathways, catalysing stepwise reactions

• They are highly specific for their substrates

• Enzymes are not used up in the process

• The majority of enzymes are proteins (globular proteins) (some exceptions such as ribosomes which are RNA not a protein)

• The native structure of the protein is critical for the activity of the enzyme

What is the classification of enzymes?

What happens in an enzyme-catalsed reaction?

• You have enzyme and substrate

• You form enzyme-substrate (ES) complex

  • Induced fit at the active site

  • Weak substrate binding, but sufficient enough to be specific

• E changes conformation to optimally bind to the transition state

  • Tighter than substrate binding

  • Lowers activation energy

• You form enzyme-product (EP) complex

  • Product is bound weakly

• You have enzyme and product

  • E releases P

  • E is not used up- It can catalyse another round and so on…

*Where old chemical bonds must break and new chemical bonds must form

How can a reaction be sped up?

• Raise the temperature (gives molecules more energy, which helps to overcome the activation energy barrier)

• Lower the activation energy (via enzyme)

• Change the pathway (mechanism) of the reaction (via enzyme)

*NOTE THE EQUILIBRIUM DOES NOT NOT NOT CHANGE, will not lead to more or less S/P (depends on environment this happens in)

Catalysed vs Uncatalysed reactions

• Activation energy has decreased from the uncat to the cat → the catalysed reaction can occur faster

• The change in free energy is unchanged (ΔG is the same - not shown here)

Modelling Catalysed and Uncat reactions on graphs - Stickase

• Enzyme needs to be complementary to the transition state not the substrate

• Try to think of this as an induced fit model

• The problem with the enzyme complementary to the substrate is that you now lock yourself in the ES complex, where the connection between the enzyme and substrate is way too strong and thus requires way more activation energy (free energy change) to enter the transition state - hence the reaction rate is very slow and you can see this on the graphs

• Note how the graphs ends and starts at the same points (the ground state of S and P is the same)

What are some Enzyme Strategies?

• Transition state stabilisation — lowers activation energy

• Specific binding — allows discrimination of correct substrate

• Induced fit — stabilised formation of transition state

• Entropy reduction — brings molecules in close proximity

• Desolvation — can make functional groups more reactive

• Acid-base catalysis — functional groups that can protonate or deprotonate substrate

• Covalent Catalysis — temporary formation of covalent bonds

How does Binding interactions drive enzyme reaction?

• Weak binding interactions between enzyme and substrate provides driving force for catalysis, especially those formed in the transition state

• Such interactions also drive specificity of enzyme reactions

What are some amino acids that often play a role in enzyme active sites?

What is a Cofactor and Coenzymes?

• A cofactor is one or more inorganic ions (non-protein) that are involved in the active site reaction. For example, Fe²⁺, Mg²⁺, Zn³⁺.

• A coenzyme is a larger organic/metalloorganic molecule that acts as a transient carrier of a functional group necessary for the enzymatic reaction.

  • For example, flavin adenine dinucleotide (FAD) that transfers electrons

  • A lot of coenzymes derive from vitamins

  • They temporarily bind to the enzyme and helps transfer atoms or electrons.

*All coenzymes are cofactors, but not all cofactors are coenzymes

What is a Prosthetic group?

A prosthetic group is a coenzyme or metal ion that is covalently bound (sometimes they can be very tightly associated if not covalently bound) to the enzyme itself.

Biotin, derived from vitamin B₇, is often found as a prosthetic group; for example, acetyl-coA carboxylase contains covalently-bound biotin (this is a key enzyme in fatty acid synthesis).

How to quantify enzyme kinetics?

• Plateaus out as substrate is all used up

Why is it important to understand Enzyme kinetics?

• It gives us an understanding of how enzymes work

• Helps us compare between different enzyme

• It allows a prediction of rates of reactions

• It provides a means to better engineer enzymes with increased/modulated activity

Graphing V0 against [S]

• Beware of units

• Velocity eventually reaches a max, where all enzyme active sites as used up… cannot theoretically go faster

• Vmax is an asymptote, the line will never touch it

• Vmax is always proportional to enzyme conc, is we use half enzyme conc, Vmax will also be halved

What are some important terms relating to enzyme kinetics?

• V₀ -- the initial velocity of a reaction at a given substrate concentration. It has units of concentration of product per reaction time.

• V_max -- the maximal velocity for a reaction at a given concentration of enzyme. It has the same units as V₀.

• K_m -- the Michaelis constant. This is the substrate concentration that results in a reaction rate equal to one-half V_max. K_m is a constant. It has units of substrate concentration.

• K_cat -- a new term I'm introducing now. This is the maximal rate for a given molecule of enzyme at saturation with substrate; it represents the number of substrate molecules converted into product in a given unit of time. Think of a single molecule of enzyme swimming in a vast sea of substrate. K_cat is constant. It's sometimes referred to as an enzyme's turnover rate. It has units of "per time".

How do the terms relating to enzyme kinetics change

• V₀ -- changes with [S], but eventually begins to plateau as rate approaches V_max.

• V_max -- this is proportional to the concentration of enzyme (i.e. [E]). So if you keep throwing in substrate but maintain [E], you'll approach V_max. But if you then double the amount of enzyme then you'll double the theoretical V_max. So, V_max scales with [E].

• K_m -- it's constant! It doesn't change. It's not dependent on [E].

• K_cat -- can be calculated if you know V_max and enzyme concentration. So, it's independent of [E] because K_cat is a measure of the rate of a single molecule of enzyme.

What is the Km Constant?

• Km → Michaelis Constant, it is defined by an equation, conceptually is the amount of substrate that gives half Vmax (the substrate concentration at which V₀ is one-half V_max)

• V0 = Vmax[S]/Km+[S]

• K_m is constant! It doesn't change with enzyme concentration

• A lower K_m essentially means the enzyme is more efficient. (Sometimes you might see K_m referred to as a measurement of enzyme affinity -- i.e. the strength of binding -- for its substrate, but this isn't strictly true.)

• So, a lower K_m means the enzyme requires less substrate to achieve a given reaction rate compared to enzymes with a higher K_m

Plotting 1/V0 against 1/[S]

• Double reciprocal plot, more technically known as a Lineweaver-Burk plot

• Super useful

• Y-axis intercept → 1/Vmax

  • If the y-axis intercept value was 0.01, then V_max = 1/0.01 = 100 µM per min

• X-intercept → -(1/Km)

  • If the x-axis intercept value was -0.5, then K_m = -(1/-0.5) = 2 mM

• Slope → Km/Vmax

*The units are given as one OVER whatever the original units were

What is enzyme inhibition?

• The action of enzymes can be inhibited

• Two broad classes of inhibition: irreversible and reversible

  • Irreversible – results in permanently non-reactive form of enzymes (often via covalent binding of inhibitor) (stops reaction) (e.g. Aspirin)

  • Reversible – inhibitor binds reversibly; action of enzyme can be restored after removal of inhibitor (slows down reaction)

• Many pharmaceutical agents are enzyme inhibitors

Competitive inhibitor

• Inhibitor competes with substrate and binds to the ACTIVE SITE

• Statins that reduce cholesterol are considered a Reversible Competitive inhibitor

Uncompetitive inhibition

• Inhibitor binds to separate site on ONLY the enzyme-substrate complex

Mixed inhibition

• Inhibitor binds to separate site on either the enzyme or enzyme-substrate complex

Competitive Inhibitor graphs

• Remember, in competitive inhibition, the inhibitor (I) is competing with the substrate. Shovel enough substrate in and you'll eventually out-compete the inhibitor

• Thus, V_max doesn't change. We can easily see that in the graph because the lines all intersect the y-axis at the same point

• However, because you need more substrate to achieve V_max, the apparent K_m (denoted αK_m) changes

• Note that as the concentration of inhibitor increases, the x-axis intercept shifts to the right; because this represents -1/Km then this means that shifting to the right results in a larger K_m

• Plug in some entirely theoretical numbers to check things:

  • Without inhibitor, if the x-axis intercept was -2 (assume the units are 1/µM as per the figure), then that means the Km would be = -(1/-2) = 0.5 µM

  • With inhibitor, if the x-axis intercept was -0.25 (i.e. shifted to the right along the axis), then that means the apparent Km would be = -(1/0.25) = 4 µM

  • Therefore, the apparent K_m increases as [I] increases

Uncompetitive inhibition graphs

• The bottom line represents the situation without inhibitor, then the two lines above it are with increasing inhibitor concentration. Note that both the y-intercept and x-intercept change with increasing [I]

• This means that V_max is decreasing with increasing [I]. (The y-axis is 1/V_max; therefore a larger V_max results in a small value on the y-axis.)

• At the same time, the x-axis intercept is shifting left, towards a more negative value. This means that the apparent K_m is decreasing.

• Think about why we see this pattern for an uncompetitive inhibitor: the inhibitor binds the ES complex and so you can think of it as removing free enzyme from the pool

• With less enzyme around, the apparent V_max decreases (recall how V_max is dependent upon enzyme concentration)

• As a consequence, the [S] required to reach half V_max (i.e. K_m) declines

  • Also think about the km decreasing because as you remove the ES from the equation, the reactions shifts to the right (to maintain equilibrium) and therefore the affinity between E and S increases and thus why Km actually decreases

Mixed Inhibition graphs

• Apparent V_max is decreasing (the y-axis intercept is increasing)

• In the image shown, apparent K_m is increasing (the x-axis intercept is increasing), but for these types of inhibitors it can actually go one way or the other, depending on the precise action occurring

• Why is this the case? Recall that a mixed inhibitor is capable of binding both the free enzyme as well as the ES complex. For V_max the situation is as for uncompetitive inhibition, where some fraction of [E] is removed entirely due to the inhibitor binding the ES complex

• For K_m the situation is complicated by whether the inhibitor has a higher affinity for the E or ES complex. In the former, it would be like the competitive example, where more substrate is required to compensate and thus the apparent K_m increases

• If the inhibitor has a higher affinity for the ES complex then it will decrease K_m as per the uncompetitive example shown before.

What are Regulatory Enzymes?

• A lot of enzymes are part of sequential pathways:

  • Reactions of one enzyme produce products that become substrates for the next enzyme

• This is especially prevalent in metabolism

• Lots of metabolic pathways have key steps that are regulated by one or more regulatory enzymes

• Allosteric enzymes are one type of regulatory enzyme

What are Allosteric Enzymes?

• Multi-domain or quaternary structure

• Involve reversible, non-covalent binding of regulatory compounds called allosteric modulators (“allosteric effectors”)

  • These can even be the substrate/product themselves

  • Binding occurs at a separate site (allosteric site)

• Binding of modulator induces conformational change that affects substrate binding (either positively or negatively)

• Allosteric can BOTH increase or decrease the rate of reaction (whereas inhibitors only slow or stop reactions)

Allosteric Enzyme Kinetics (negative or positive modulator)

• Behavior doesn’t obey Michaelis-Menten kinetics

• Besides is an example for when the substrate itself is a positive modulator of activity

• High-activity R state → Relaxed

• Low-activity T state → Tense, looking to bind

• K0.5 changes

• Km is not given

What are Isozymes?

• Isozymes are enzymes that catalyse the same reaction but have different primary structures (they’re also encoded by different genes)

• A good example of this is hexokinase, which catalyses the phosphorylation of glucose to glucose-6-phosphate

Isozymes - Hexokinase Example

What is Metabolism?

•  Refers to all the reactions that occur in our body that provide the body with energy to carry out all its bodily processes

• Metabolism is defined as the sum of all the chemical reactions taking place in a cell or organism that allows life to be sustained. This highly coordinated activity and includes many reaction pathways, includes:

  • Obtaining chemical energy from macromolecules

  • Converting nutrient molecules to cellspecific molecules

  • Synthesising macromolecules from precursor molecules

  • Synthesising and degrading cell-specific molecules

• A chemical reaction occurs when atoms have enough energy to combine or change bonding partners

Metabolism and Energy

• Metabolic reactions involve energy changes

• Energy is the defined as the capacity to do work or the capacity for change

• In Biochemical reactions, energy; is the capacity for change

• Energy changes are usually associated with changes in the chemical composition and properties of molecules

What are the types of Energy?

• All forms of energy are either:

  • Potential energy—energy stored as chemical bonds, concentration gradient, charge imbalance

  • Kinetic energy—the energy of movement

• Energy can be converted from one form to another

What is Bioenergetics?

Is the quantitative study of energy transductions (the changes of one form of energy to another)

The law of thermodynamics (overview)

The laws of thermodynamics apply to all matter and all energy-transforming reactions

1st Law of Thermodynamics

Energy is neither created nor destroyed

• When energy is converted from one form to another, the total amount of energy before and after the conversion is the same

2nd Law of Thermodynamics

When energy is converted from one form to another, some of that energy becomes unavailable to do work

• No energy transformation is 100% efficient, some energy is unavailable (due to disorder or entropy)

What is Entropy?

• Entropy is a measure of the disorder in a system

• It takes energy to impose order on a system. Unless energy is applied to a system, it will be randomly arranged or disordered

• During physical exercise, muscles convert chemical energy (from food) to mechanical energy (contraction)

• Not all of this energy is available to do work, some of it is released as heat energy due to entropy

What is Free Energy?

• Free energy is a measure of the capacity of a system to do work

• We can get a negative, positive or 0 answer this can tell us the type of reaction

What are the types of energy conversions?

• If ΔG is –ve, free energy is released (EXERGONIC)

• If ΔG is +ve, free energy is required (ENDERGONIC)

• If free energy is not available, the reaction does not occur

• If ΔG = 0, the reaction is at chemical equilibrium – A <=>B (Concentrations of A and B will determine the direction of the reaction)

What are coupled reactions?

• Many biological reactions are thermodynamically unfavourable (require energy or are endergonic)

• To overcome this, endergonic reactions are coupled with exergonic (or energyreleasing) reactions—to do biological work

• The reactions of metabolism harness energy-releasing reactions to drive energy-requiring reactions

What is Anabolism and Cataboilism

ANABOLISM

• Uses precursor molecules to synthesise complex macromolecules

• Requires an input of energy

• Energy is captured in chemical bonds and stored as potential energy

CATABOLISM

• Breakdown of complex molecules

• Release of energy stored in chemical bonds

• Occurs via oxidative pathways

• Released energy is recaptured in new chemical bonds

Example of Chemical Coupling

• Chemical coupling of exergonic and endergonic reactions allows otherwise unfavourable reactions to take place.

• The “high-energy ” molecule (ATP) reacts directly with the metabolite that needs “activation.”

• The final reaction will be: Glucose + ATP → GLU 6-P + ADP (deltaG = 13.8 + (-30.5) = -ve

  • The energy requiring reaction harnesses the energy from the energy releasing reaction

What is the structure of ATP (adenosine Triphosphate)

• ATP is considered the energy currency, because the hydrolysis of the phosphate groups release energy

How do electrons work again

• Capturing ELECTRONS is the key to harnessing energy

• A substance that loses electrons is OXIDISED

• A substance that accepts electrons is REDUCED

• AN OIL RIG CAT

Biological Oxidation-Reduction Reactions

Electrons are transferred from one molecule (electron donor) to another (electron acceptor) in one of 4 ways in biological systems

• Directly as electrons

  • Fe²⁺ + Cu²⁺ → Fe³⁺ + Cu⁺

• As hydrogen atoms (H+ + e-) (FAD)

• Transfer as a hydride ion (which is made of a 2e- and 1H+) (NAD)

• Direct combination with oxygen

  • Oxygen combines with an organic reductant and is covalently incorporated to the product

  • R-CH3 + 1/2O2 → R-CH2-OH (oxidation of a hydrocarbon to an alcohol)

What are the most common electron carriers/acceptors?

• NAD accepts e- and will be reduced to NADH

• FAD will accept 2e- and will be reduced to FADH₂

• The molecules in orange are the electron carriers

NAD+/NADH as Electron Carriers in Redox Reactions

• O2 accepts electrons from NADH:

  • NADH + H⁺ + ½ O₂ → NAD⁺ + H2O

  • The reaction is exergonic: ΔG = –52.4 kcal/mol

  • O2 is the oxidizing agent, reduced to water

  • Spontaneously occurring energy producing reaction

FAD/FADH2 as Electron Carriers in Redox Reactions

• O2 accepts electrons from FADH2 and is reduced to water

• The reaction is exergonic: ΔG = –40 kcal/mol

• Spontaneously occurring energy producing reaction

What does the transfer of electrons to oxygen depend on?

• The transfer of electrons to oxygen depends on:

• SPECIFIC CARRIERS in the RESPIRATORY CHAIN or THE ELECTRON TRANSPORT CHAIN

• REDOX POTENTIAL

What is Redox Potential?

• When two conjugate redox pairs are in solution;

  • The transfer of electrons from the donor to the acceptor depends on the relative affinity of the electron acceptor for the electrons (look in the image at the way the e- move)

  • Electrons always “fall” toward the species with the highest (most positive) reduction potential, because that species is the strongest oxidant — the best electron acceptor.

  • A reductant is the opposite: it is an electron donor. Electrons are not attracted to donors; donors push electrons away.

• Free energy exchange in oxido-reduction reactions is proportionate to the ability of reactants to donate or accept electrons

• ΔG can be expressed as an oxidation-reduction or redox potential E⁰

What are the main structures involved in electron transport

• Complex I: NADH dehydrogenase

• Complex II: Succinate dehydrogenase

• Complex III: Ubiquinone:cytochrome c oxidoreductase

• Complex IV: Cytochrome oxidase

• Q: Coenzyme Q

• Cyt c: Cytochrome C

Where is the electron transport chain located?

• The electron transport chain is located in the inner mitochondrial membrane

• In the table above, we can see the transfer of electrons from the electronegative to the electropositive, indicated how the molecules at the top can readily donate e- and how oxygen at the bottom is a strong acceptor of electrons

Electron Transport Chain — Complex I

• NADH dehydrogenase complex

• Multi subunit complex (around 45)

• Accepts electrons from NADH

• Electrons are transferred to coenzyme Q (quinone), which accepts these e- and will be reduced to QH2 (Ubiquinone)

• QH2 is a mobile carrier (moves freely in the inner membrane to Complex III)

• As electrons move from one carrier to the next, there is a release of free energy, and therefore that free energy means there is sufficient energy in the cell to expel 4 protons from the matrix to the intermembrane space

• Every 2e- from NADH → EXPELS 4 PROTONS

Electron Transport Chain — Complex II

• Succinate dehydrogenase

• Accepts electrons from FADH₂ (from the Krebs cycle)

• Passes electrons to Coenzyme Q (quinone), which accepts these e- and will be reduced to QH2 (Ubiquinone) which will carry electrons to Complex III

• Single enzyme with dual roles:

  • Citric acid cycle (later)

  • Capture and donate electrons in the ETC

• DOES NOT EXPEL PROTONS

Electron Transport Chain — Complex III

• Cytochrome C oxidoreductase

• Receives electrons from QH2

• Electrons are transferred to Cyt c (mobile carrier in the intermembrane space)

• There is a transfer of electron, which means there is free energy released which is sufficient enough to EXPELS 4 PROTONS

Electron Transport Chain — Complex IV

• Cytochrome oxidase

• Accepts electrons from cytochrome c

• Electrons are transferred to oxygen

• O₂ + 4H⁺ → 2H₂O (oxygen is reduced thus is the oxidizing agent at the end)

  • The final electron acceptor station

• Release of free energy → EXPELS 2 PROTONS

ETC Summery

• The complexes are found embedded in the inner mitochondrial membrane

• Take a look at BOTH equations

What does it mean for a proton gradient to be established?

The resulting proton gradient causes… (potential energy)

• Chemical potential energy produced by the pH gradient

  • Matrix pH 8

  • Outside pH 7

• Also generates an Electrical potential energy (Voltage gradient)

  • The matrix is electronegative compared with the outside space

What is the chemiosmotic model for ATP synthesis

• The energy released from the transfer of electrons (from NADH and FADH2 formed as a result of biological oxidation reactions) is stored as a transmembrane difference in proton concentration. This drives ATP synthesis.

• Chemical potential + electrical potential → ATP synthesis driven by proton motive force

• Complex V - ATP synthase complex

Complex V - ATP synthase complex

• Experimental values show;

  • 4 protons = synthesise ONE ATP

• Made up of 2 subunits F_o (oligomycin - an antibiotic) and F₁ (has catalytic activity, responsible for the phosphorylation of ADP to ATP)

• Acts as a channel to allow the reentry of protons back into the matrix

• The rotation of F_o subunit as H+ load in causes a confirmational change to F1 → this conformation change activates the enzyme ATP synthase → once activated will phosphorylate ADP to form ATP

How many ATPs are synthesised when NADH and FADH2 are oxidised?

• 4 protons = synthesise ONE ATP

• We know that the transfer of electrons from NADH to oxygen ∆G = -190 kJ/mol, which is a HUGE energy release

• ATP synthesis is endergonic (requires energy): ADP + Pi → ATP ∆G = +52 kJ/mol

  • But luckily it offsets the transfer of electrons from NADH to oxygen

What happens to the ATP that is synthesized?

• Proton Motive Force drives ATP synthesis as well as transports substrates into the matrix and products out of the matrix

• Inner membrane space is very impermeable, then how does it get all the substrates to synthesis ATP and what happens to the ATP

  • ADP comes from outside and is transported by the Adenine nucleotide translocase (antiporter) which is a transport protein

  • The phosphate (in the form of dihydrogen phosphate) comes from outside and is transported by a phosphate translocase (symporter) which is a transport protein

  • Now substrates are in the matrix for the phosphorylation to happen

  • The ATP synthesized is then transported out via the Adenine nucleotide translocase (antiporter)

    • The Adenine nucleotide translocase brings a charge of -3 inside (ADP) and then releases a -4 charge (ATP) outside, since the outside is more positive the transport of ATP (the more negative one) is favored

What is Oxidative Phosphorylation?

• The process by which the oxidation of NADH or FADH2 (transfer of electrons to oxygen) results in the synthesis of ATP

The electron transport chain and ATP synthesis are COUPLED

• O2 consumed → reduction of O2 to H2O (GRAPH UP)

• At the start both graphs are flat since not much O2 is being consumed and not much ATP is being synthesized

• Then they added succinate → this oxidizes to fumarate, during this reaction FAD is reduced to FADH2, FADH2 then passes its electrons to the reaction

• Now the O2 consumed (reduced) and ATP synthesis is steadily increasing - means electrons are being transported now

• Add CN- (cyanide)

  • This inhibits complex IV, this means the electrons are not transported to oxygen, ATP synthesis is also come to a halt (plateau)

• THIS SHOWS US that if the ETC is inhibited, ATP synthase is also inhibited and thus these processes are COUPLED

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• Succinate added, adds electrons, but the graphs shows that O2 is not reduced as much and ATP is not synthesized as much (naturally as there is no substrate)

• Thus now add the substrates ADP and Pi, now with BOTH the substrates and electrons, more O2 is consumed and as a result more ATP is synthesized

• Now add Venturicidin or oligomycin to inhibit ATP synthesis → now we see ATP synthesis comes to a halt as a result the ETC also comes to a halt

• THIS SHOWS US that if the ATP synthase is inhibited, the ETC is also inhibited and thus these processes are COUPLED, INTACT mitochondria

• Now uncouple the process by adding DNP (an uncoupler), then we see ATP synthesis continue to be inhibited, but ETC can continue to take place (not having to occur at the same time)

The electron transport chain and ATP synthesis can be UNCOUPLED, HOW?

Due to…

• Mechanical damage

  • Leakages → hard to establish a PMF

• Chemical reagents (e.g. 2,4-dinitrophenol (DNP))

  • DNP is a hydrophilic proton carrier (uncoupler), therefore it can diffuse into the matrix, carrying those protons which will then dissipate the proton gradient and PMF that had been established, now ATP synthesis does not occur, only the ETC can take place thus why it is an uncoupler

• Natural uncouplers

  • Commonly found in newborns

  • Uncoupling protein 1

  • Thermogenin found in brown adipose tissue - which has a brown color due to an excess of mitochondria which therefore has a high conc of cytochrome C

  • Again provides a channel for protons to enter the matrix, thus dissipates the protons gradient and PMF, therefore ATP is not produced BUT the energy produced is released as heat energy which is important to help new borns/animals to adjust to the temp of the environment

Whoa are the inhibitors of the electron transport chain and ATP synthesis?

• Venturicidin and Oligomycin (inhibit the Fo complex of the ATP synthase)

• Look at all the ones given in the image above (these inhibit the ETC)

• THESE CEASE PRODUCTION - doesn’t decrease it

How is oxidative phosphorylation controlled?

• ATP synthesis is regulated by the energy needs of the cell

• Depends on the mass action ratio [ATP] / [ADP][Pi] (product/substrate)

• High energy energy, lots of ATP, low ADP → respiration is resting

• ATP decrease, ADP increase → increase in respiration, coupled with oxidative phosphorylation, mass action ratio is low

• ATP and ADP back to normal → respiration resting

What is Acetyl CoA?

• Make up of Coenzyme A + Acetate - has 2 Carbon atoms

• Coenzyme A is derived from pantothenic acid (part of the vitamin B group) and has a reactive thiol group (-SH) which condensed with the acetate to form Acetyl CoA (by forming a thiol ester linkage, which is very important because it is the hydrolysis of this bond in Acetyl CoA that releases a HUGE amount of energy (-31.4 KJ/mol) which is sufficient to dive the ENTIRE CAC)

Where does the Citric Acid Cycle (CAC) take place?

• In the mitochondrial Matrix

• The 2 carbon atoms in Acetyl CoA are released as CO2 through multiple reduction/oxidation reactions, these electrons released are captured to form the NADH, FADH2 and GTP

• The citric acid cycle is aerobic and therefore cannot occur in the absence of oxygen

  • Even though oxygen is not used directly in the cycle, it is required indirectly because NADH and FADH2 must be recycled, This regeneration happens only in the ETC which can only run if oxygen is present as the final electron acceptor

CAC: Step 1 Condensation

• 2C Acetyl CoA + 4C Oxaloacetate → 6C Citrate

• The energy to carry out the reaction is provided by the hydrolysis of the thiol ester linkage in Acetyl CoA and it provides a huge surplus of energy as seen with the DG value given