BMS1041 Biochemistry

What effect do proteins have on cells

All cells contain the same genome but it is expressed differently via proteins

What are proteins made of

Amino acids, which are broken down into ą carbon, carboxyl group, amino group and a side chain (R group)

What are the different classifications of amino acids

Non polar aliphatic R groups, Aromatic R groups, Polar uncharged R groups, Positively charged R groups, Negatively charged R groups

Describe the peptide bonds in bonding amino acids

Amino acids can be covalently linked by a peptide bonds in

The hydroxyl group of one AA is bound to the amino terminal group of another. This releases one molecule of water (condensation reaction)

What is an oligopeptide

A few amino acids joined together

How are polypeptides read

From left to right (amine terminal to carboxyl terminal)

How can proteins be quantified in solution

Proteins contains amino acids with aromatic groups can absorb UV light (lambert beet law) this can be exploited by researchers to characterise proteins in a solution

What is molecular weight normally measured in

Kilodaltons

What does oligomeric mean

If a protein contains two identical polypeptides (these peptides are said to be protomers)

What is the prosthetic group

The part of a substance that isn't an amino acid e.g. the lipid part of a lipoprotein

Describe the 4 levels of protein structure

Primary protein structure - sequence of a chain of amino acids

Secondary protein structure - local folding of the polypeptide chain into helices or sheets

Tertiary protein structure - 3d folding pattern of a protein due to side chain interactions

Quaternary protein structure - protein consisting of more than one amino acid chain

Describe the primary structure of a protein

A linear sequence of amino acids. Peptide bonds are formed between amino acids. It is formed during translation in the cell cytoplasm. It can then be modified by post translational modifications

Describe the constraints put on the structure of a protein due to the covalent peptide bond

In a polypeptide the the ą carbons of adjacent amino acids are separated by covalent bonds. 6 atoms of the peptide group lie in a straight plane. The peptide bond is rigid and planar meaning the only flexibility is between the phi and psi bonds

Describe the secondary structure of proteins

The folding of the peptide chains into a helices, beta sheets or beta turns. Hydrogen bonds are formed and the secondary structure forms globular or fibrous proteins

Describe an alpha helix

It is generated when a single polypeptide twits around itself. There is a formation of hydrogen bonds between each 4th peptide bond ( doesn't involve the R group)

They are mostly right handed twists

There is a turn every 3.6 amino acids

What amino acids are important to note with alpha helix's

Alanine has the nuggets tendency to form a helices due to its structure

Proline is found outside the alpha helix as it doesn't allow for the flexibility

Describe beta strands and beta sheets

More common in globular proteins

They can be parallel or anti parallel depending on direction

Flexible loops and turns link regions of secondary structure

Hydrogen bonds form between peptides in different strands, the bonding is stronger in anti parallel beta sheets because C=O and N-H groups are better aligned

Beta turns are common in globular proteins

What is circular dichroism spectroscopy

Any form of structural asymmetry in a molecule gives rise to differences in absorption of polarised light, circular dichroism spectroscopy is the measurement of this difference

Describe tertiary structure

The 3d conformation of a protein. It is maintained by disulfite bonds, salt bridges and hydrogen bonds.

Proteins can be fibrous, globular, membrane or intrinsically disordered

What are the weak interactions that stabilise the 3d conformation of a protein

The hydrophobic effect is the most important weak interaction - causes the hydrophobic amino acids to hide inside the hydrophilic outer side

Other weak forces include van der waals, electrostatic attraction and hydrogen bonds

What are the characteristics of fibrous proteins and give examples

Provide support, shape and external protection

Alpha keratin of hair, Silk fibroin, Collagen of tendons

Describe the structure of Alpha keratin

It is a Fibrous protein, made of alpha helixes cross linked by disulfide bonds

2 alpha helixes coil together to form protofilament which then forms protofibril

Very strong protein - found in hair

Describe the structure of collagen

Is a fibrous proteins.

3 helically intertwined alpha helixes

Genetic mutation in collagen cause osteogenesis imperfecta and Marfan syndrome

Describe the structure of silk fibroin

A fibrous protein with beta confirmation.

It is made of anti parallel beta sheets. This confirmation is highly extended so does not allow for stretch. The structure is flexible because the sheets are kept together by weak interactions

Describe globular proteins

Roughly spherical in shape and might have several types of secondary structures folded together

Often soluble as hydrophobic residues are hidden inside by the hydrophilic outside (this is the most stable structure)

What is a motif

specific arrangement of several secondary structure elements

Describe the structure and function of myoglobin

Function - to store oxygen and facilitate oxygen diffusion in rapidly contracting muscle

Globular protein

A single polypeptide chain with a single iron protoporphyrin or Haem group

The backbone is alpha helixes and beta turns

So compact that it only has room for 4 molecules of water

Describe the structure of haem

Consists of N organic ring structure (protoporphyrin IX) with a bound atom in its ferrous (Fe2+) state

How do proteins interact with other molecules

A ligand can bind to a protein at a binding site that is complimentary to the ligand

This may cause conformational changes in a protein e.g. the induced fit

Proteins that bind to ligands also normally release ligands

Proteins that bind and chemically alter molecules are called enzymes

The relationship between unbound protein and unbound ligand to bound protein ligand can be described with the association constant Ka

How does oxygen bind to Haem

It is an exponential growth. The binding increases as the substrate concentration increases.

What is quarternary structure

When proteins have two or more separated polypeptide chains. It is formed by the assembly of individual polypeptides into a larger functional cluster. Often have a regulatory role or a structural role

What is the structure of Hemoglobin

2 alpha and 2 beta chains. It has the Haem group (the prosthetic group). As it has a prosthetic group it is called a conjugated protein. It transitions between 2 major confirmations T sate and R state

Describe the 2 different states of Haemoglobin

2 major confirmations R state and T state.

When there is no oxygen bound the most stable conformation is the T state

When oxygen binds to the T state it triggers a conformational change to the R state

How many oxygen molecules bind to Hemoglobin and why?

4 oxygens can bind to Oxygen, one per subunit.

The more molecules of O2 that Hemoglobin binds to the higher its affinity for oxygen becomes

Why does Haemoglobin bind oxygen cooperatively?

Because it has to bind efficiently in the lungs where the partial pressure of oxygen is high and release oxygen into tissues where the partial pressure is low

Haemoglobin transitions from low to high affinity states as more oxygen molecules are bound to

How does Haemoglobin transport CO2 and H

It transports it from tissues to the lungs and kidneys

The CO2 is hydrated to form bicarbonate in a reaction catalysed by carbonic anhydrase in the erythrocytes

The reaction also increases the concentration of H+ in the tissues

At the relatively low pH and high CO2 concentration H+ and CO2 are bound and affinity for O2 decreases

In the lungs CO2 is secreted and blood pH rises. Therefore affinity to O2 increases

What is the blood pH and tissue pH and oxygen binding pH

Blood pH - 7.6

Experimental binding of O2 to Haemoglobin - 7.4

Tissue pH - 7.2

How is oxygen binding to Hemoglobin regulated and what changes are made at altitude

It is regulated by 2,3 beta glycerophosphate which binds to Haemoglobin and regulates the affinity of Hemoglobin for Oxygen.

It is important for the adaption to high altitudes where partial pressure is low

After a few hours at high altitude the BPG concentration in blood begins to rise leading to a decrease in affinity for oxygen - increases release in peripheral tissues

Give an example of quarternaru structures

Viral capsids

What diseases are caused by an accumulation of misfolded proteins

Alzheimers disease and Parkinsons

What are the steps of X-ray Crystallography for determining protein structure

1. Protein purification

2. Crystallise the protein

3. Collect diffraction data

4. Calculate electron density

5. Fit known amino acids residues into density

What are the pros and cons of X-ray crystallography

It's a well established technique, it has no size limit

Difficult to use for membrane proteins

What do Enzymes act on

Substrates

Describe the structure of an enzyme

Binding site in the centre protected by hydrophobic amino acids

What is Chymotrypsin

A protease (enzyme) that catalyses the hydrolysis of peptide bonds. It is specific for peptide bonds next to aromatic amino acids

Describe induced fit

Conformational changes may occur upon ligand binding

Allows for tighter and higher affinity binding of the ligand

What is the point of enzymes in the body

Releases nutrients that make up our bodies and energy we need to exercise. Enzymes also catalyse all reactions in cells

What is the importance of enzymes in industry

Rennet in cheese is a mix of enzymes

Invertase breaks down hard sugars into soft sugars

Phytases are added to animal feed to make minerals more bioavailable

Why are enzymes necessary

Most biological reactions are chemically infavourable

What are riboenzymes

RNA molecules that have a complex tertiary structure that makes it catalytically active

What is a cofactor and coenzymes

extra molecules needed for enzymes to function

commonly are metal ions like Fe2+ and zinc

Whats the difference between cofactors and coenzymes

More complex organic cofactors are called coenzymes

Describe coenzymes

complex organic molecules needed for enzyme function

They are often derived from vitamins

Often acts as transient carriers for functional groups

What enzymes need both a coenzyme and a metal cofactor

Alcohol dehydrogenase - coenzyme NAD+ converts to NADH (also has zinc)

What are prosthetic groups

they bind tightly or covalently to a protein and have a structural element. Co enzymes that bind more loosely

What is a Holoenzyme vs an apoenzyme

Apoenzyme - inactive protein portion

Holoenzyme - whole enzyme and coenzyme complex (is active)

How do enzymes affect a reaction

Increase rate of reaction but not its equilibrium

(makes P quicker but doesn't make more of it)

How do enzymes lower activation energy

Intermediates are introduced

the activation energies required to produce these intermediates is lower and the transition state is at a lower energy level so rate of reaction increases

What are metalloproteins

Proteins that include a metal in the protein structure

What are the roles of the metal within a metalloprotein

Binds and orientates ligands and substrates

Mediate oxidation-reduction reactions

Charge stabilization

Promote nucleophilic catalysis

What happens to absorption when Haem is bound to oxygen

Deoxy Haemoglobin - 429 nm

Oxy Haemoglobin - 414nm

This explains why oxygenated blood is red but deoxygenated is blue

What are Restriction enzymes

Enzymes that are able to recognize DNA sequences in target genome and cut them

What do restriction enzymes recognize?

Invert repeats (you can read them either way exactly the same - palindromic)

What is the importance of magnesium in its metalloprotein

Only when magnesium ions are present the confirmation is tight - essential for function

Can also reduce electrostatic repulsion between 2 Aspartates

Facilitates orientation

Is haemoglobin an enzyme?

No

What is the name of an enzyme made of RNA

Ribozyme

ADH catalyses the reaction ethanol + NAD+ = Acetaldehyde + NADH. What is the substrate

Ethanol

If the concentration of products is higher than that of substrates, how is the equilibrium constant

Keq is higher than if the opposite was true

How does conversion effect how favourable a reaction is

A reaction where a lot of substrate is converted into product is more energetically favourable

Which is the rate limiting step in a multi step reaction

The step with the highest activation energy is the rate limiting step. To speed up the overall reaction the rate of this step must be increased

Some reactions may have steps with similar activation energies. These are all the rate limiting step

How do enzymes interact with substrates

Chemical reactions take place between the substrate and some of the enzymes functional groups

Transient covalent bonds may be formed or a group may be transiently transferred from substrate to enzyme

Transient non covalent interactions stabilise the ES complex

Covalent interactions between substrate and enzyme lower the activation energy

How do enzymes lower the activation energy?

Forming each weak interaction in the ES complex releases some free energy that stabilizes the interaction.

The energy of all these weak interactions added up is called the binding energy (ΔGB ).

Binding energy is used to lower the activation energy of the reaction

How do weak interactions effect induced fit?

Weak interactions are optimised in the reaction transition state that the substrate becomes as it is changing into a product. Often the enzyme also undergoes a conformational change. This is induced fit

What are other ways enzymes help reactions

Entropy reduction - Enzymes restrict movement of substrates making it more likely they will collide

Removal of solvation shell - Dissolved substances are surrounded by water molecules that may block reaction sites. A substrate binding to an enzyme usually loses its associated water molecules

Distortion of substrates - Binding energy can be used to twist substrates to the right orientation

Alignment of functional groups - they are positioned in a way that aids reaction

Why is initial velocity important

If we increase substrate concentration the reaction may react more quickly but the concentration of the substrate changes as the substrate is used up

Initial velocity is the time that the reaction is most linear

What units are used for substrate concentration

mini molar (mM)

What is Michaelis constant

The [S] when the reaction rate is half of Vmax

How is Km used as an indicator of an enzymes substrate affinity

an enzymes with a low Km has a high affinity for a substrate because it takes a lower concentration of substrate to reach 0.5Vmax

What is the Lineweaver Burk (double reciprocal) plot used for

To visualise the relationship between Km and Vmax

y axis = 1/Vmax

x axis = -1/Km

Slope = Km/Vmax

What is Vmax dependant on

Substrate concentration

Specific conditions

pH

Temperature

concentration of enzymes

Inhibitors

What is Km dependant on

pH

inhibitors

What is a turnover number

equivalent to the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate

What are 2 substrate reactions

When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms: Sequential mechanism

Ping-Pong mechanism

What is sequential mechanism

Can happen in random order or a specific order but eventually end up with the same complex or when the binding of substrate 2 is dependent on the binding of substrate 1

What is the ping pong mechanism

Substrate binds ot enzyme and makes a product which makes a change that allows the binding of substrate 2

What are the roles of metals in protein function

Bind/ orientate ligands and substrates

Mediate oxidation-reduction reactions

Charge stabilization

Promote nucleophilic catalysis

How is iron in our bodies

In our bodies iron is complexed with the porphyrin ring where its bound to 4 oxygens

What is the absorption of different amounts of haemoglobin

Iron can change absorbance when bound to oxygen

Oxy haemoglobin is less absorbent

Oxy - 414

Deoxy - 429

What is a chromophore and give an example

molecule which absorbs light at a particular wavelength and reflects colour as a result.

the heme group is a strong chromophore

What is a Soret band

an intense peak in the blue wavelength region of the visible spectrum

How does oxygen alter the position of the soret band

Oxygen binding alters the electronic properties of the heme and shifts the position of the soret band 414nm

This can be monitored by UV-Vis spectrophotometry

Give examples of metalloproteins

Restriction enzymes, Haemoglobin, Carboxyl glutamate & calcium, Alcohol dehydrogenase (ADH) and zinc

What are restriction enzymes

an enzyme that has the property of cleaving DNA molecules at or near a specific sequence of bases - produced by certain bacteria

How are restriction enzymes structured to allow them to be functional

The enzymes must be in dimeric form has 2 Mg2+ binding sites

Complex is only bound tightly when two magnesium are bound - when bound they interact with 2 aspartate residues in the active site

What is cognate DNA vs non cognate DNA

Cognate DNA: This refers to the specific DNA sequence that a restriction enzyme is designed to recognize and cut. When the enzyme binds to its cognate site, it undergoes a conformational change that enables cleavage at or near that sequence.

Non cognate DNA: an interaction that is not appropriate or specific

This refers to DNA sequences that do not match the enzyme's recognition site. Restriction enzymes can still bind to non-cognate DNA, but they do not typically undergo the conformational changes needed for cleavage. Instead, they often exhibit non-specific interactions with the DNA and slide along it to locate their cognate site.

Why is magnesium important for enzyme activity

Many enzymes that act on phosphate containing substrates require Mg2+ or some other divalent cation for activity

How does Mg2+ allow restriction enzymes to cleave water

Magnesium (Mg²⁺) is held in place:

The enzyme holds the Mg²⁺ ion using two aspartate residues (amino acids in the enzyme) and one oxygen atom from the DNA's phosphate group.

Magnesium helps position a water molecule:

The Mg²⁺ ion grabs onto a nearby water molecule, holding it in the right place.

Magnesium helps activate the water:

The Mg²⁺ ion makes the water molecule more reactive, turning it into a strong attacker.

The water cuts the DNA:

The activated water molecule attacks the phosphorus atom in the DNA backbone, breaking the bond and cutting the DNA at the target site.

What is a Scissile bond

a covalent chemical bond that can be broken by an enzyme

What is the difference in structure with a gamma carboxyglutamate vs a normal carboxyglutamate and what difference does this give to its structure

Gamma carboxyl glutamate has an extra carboxylate.

This provides it with the ability to bind to calcium ions

This is important in blood coagulation as it allows the enzyme to be bound to phospholipids to aid coagulation

Where do we get vitamin K from in our diet

mainly greens