enzymes + proteins

Question 1

what are the 6 classes of amino acids?

Answer 1

Aliphatic

Aromatic

Sulphur-containing

Acidic

Basic

Polar

Question 2

Amino acids are affected by the pH levels whether an environment is acidic or alkalinic or neutral.

Describe how the pH affects them.

Answer 2

n a low pH environment = [H+] ↑ = protonated carboxylic acid = cation/acidic amino acid

In a neutral pH environment = neutral levels = both negative and positive charge in amino acid’s functional groups = zwitterion amino acid

In a high pH environment = [H+] ↓ = deprotonated carboxylic acid = anion/basic amino acid

Question 3

what are aliphatic amino acids?

Answer 3

they have an ‘R’ group that consists of hydrocarbon chains or simply a hydrogen.

The longer the hydrocarbon chain the more hydrophobic, nonpolar the amino acid is

Question 4

what are aromatic amino acids?

Answer 4

they have an ‘R’ group that contains a hydrocarbon ring

Question 5

what is phenylketonuria?

Answer 5

(PKU) is an inborn metabolic error that causes abnormal levels of an aromatic amino acid called Phenylalanine, which can cause neurological damage.

Question 6

whar are sulphur containing amino acids?

Answer 6

they contain sulphur that allows for disulfide bridges, which are covalent sulphur-sulphur bonds between two compounds.

Question 7

what is proline?

Answer 7

it’s a miscellaneous amino acid which doesn’t fit in any of the classifications of amino acids, but has an unusual ring shape that gives tissues high strength.

Question 8

what is the primary structure of a protein?

Answer 8

it’s the sequence in which amino acid monomers are covalently bonded together to form a polypeptide chain using a peptide/amide bond

Sequence of a chain of amino acids:

Peptide → Dipeptide → Tripeptide → Polypeptide

Question 8

what is the seconday structure of a protein?

Answer 8

it’s the 3D spatial arrangement of amino acids located near each other in the polypeptide chain.

It relies on hydrogen bonding between a carbonyl Oxygen of one amino acid and the amino Hydrogen of another.

Two basic types that can form: alpha helix & beta sheet, depending on the bonding between amino acids that make up the primary structure.

Question 9

what is an alpha helix and a beta sheet?

Answer 9

Alpha Helix is a secondary structure that is made up of polypeptide chains, which looks like a coil.

Beta Sheet is a secondary structure that is made up of polypeptide chains, which looks like an extended sheet.

They are both highly dependant on hydrogen bonding between a Carboxyl Oxygen and an Amino Hydrogen.

Question 9

what are the 7 functions of proteins

Answer 9

Structural proteins → collagen / keratin

Enzymatic proteins → Digestive enzyme e.g. amalyse

Receptor proteins → G proteins

Hormonal proteins → Insulin

Transport proteins → Haemoglobin / Sodium & Potassium pump

Storage proteins → Ferritin (storage protein for iron)

Defensive proteins → Immunoglobulins

Contractile proteins → Actin / Myosin in muscle fibres

Question 10

what is the quaternary structure or a protein?

Answer 10

here there are several polypeptide interactions.

e.g. haemoglobin, which contains four highly-folded polypeptide chains.

Question 10

what is denaturation?

Answer 10

Denaturation is the process through which the biological functionality of a protein is lost, when the bonds and interactions are disrupted and the protein loses its secondary and tertiary structure.

It rarely breaks the primary structure of individual peptide bonds.

Question 11

what is the tertiary stucture of a protein?

Answer 11

it results when functional groups of ‘R’ chains of amino acids in the polypeptide chain interact with one another.

Van der Waals, Ionic, Hydrogen, Disulphide bridges and Hydrophobic interactions can all be involved here.

Question 12

what is the post/ co translational modification of a tertiary structed protein?

Answer 12

where additional modification occurs to a them, e.g. adding macromolecules, contributes to them being quaternary structured proteins that are known as Conjugated proteins.

Question 13

what are 3 types of conjugated proteins?

Answer 13

1. Glycoproteins, one or more carbohydrate molecule covalently attached to a protein.
2. Lipoproteins, proteins combined with lipids.
3. Metalloproteins, protein molecules with a metal ion within their structures.

Question 14

What are the effects of Protein Glycosylation?

Answer 14

Increased Stability

Altered Solubility

Cell signalling

Orientation, new configurations of the protein.

Question 14

what is protein glycosylation?

Answer 14

a reaction in which a carbohydrate, usually an oligosaccharide, is attached to a protein, which can be a Post or Co-translational modification, to create a Glycoprotein.

Question 14

what are metalloproteins? give an example:

Answer 14

one of the conjugate proteins that are formed from a combination of protein molecules with metal ions in their structures to work as co-factors.

One famous example is haemoglobin which is used to transport oxygen in the blood.

It is a Large quaternary structure, made from 4 polypeptide chains comprised of 2 alpha subunits and 2 beta subunits.

Each subunit has an organic molecule called Haem which contains an atom of Iron.

Each haemoglobin molecule with its four subunits can carry four molecules of oxygen.

Question 15

what are lipoproteins?

Answer 15

They are used to facilitate the transport of hydrophobic lipids.

A group of them can form complexes called Apolipoproteins used to transport lipids in the blood and cerebrospinal fluid.

Question 16

How is Glycoprotein HbA1c used to detect Diabetes Milletus ?

Answer 16

Glycoprotein acts as a marker for the immune system cells to recognise self vs non self cells

The immune system secretes antibodies as an immune response against foreign bodies.

Detection of the concentration of glycoproteins HbA1c in the blood of a diabetic person.

People who are undiagnosed but afflicted by Diabetes Milletus have a chronic high level of glucose which binds to haemoglobin over time forming Glycoproteins.

This can be used by clinicians to detect the glycoproteins in the blood to diagnose a person.

Question 16

what are the 3 classifications of proteins based on their function?

Answer 16

1. Globular Proteins
   - Storage
   - Enzymes
   - Hormones
   - Transporters
   - Structural

e.g. Immunoglobulins & Growth Hormone

They can be used clinically to check for pathology e.g. high levels of proteins in blood

2. Fibrous Proteins
   e.g. Muscle Fibres & Connective tissue

• Collagen for stability and tensile strength
• Vitamin C is an important precursor and co-factor to produce collagen.

3. Membranous Proteins
   - Membrane transporters and enzymes, Cell adhesion molecules

• Used for transportation and cell signalling

Question 17

what is sickle cell anaemia?

Answer 17

it’s an inherited blood disorder where a single nucleotide substitution leads to a pathological phenotype by changing the expressed amino acid thus changing the structure of the protein.

Adenine to Thymine substitution leads to → encoding changes from Glutamate to Valine

In a Homozygous individual (containing two abnormal alleles):

Uniform biconcave shaped erythrocytes → Rigid sickle shaped erythrocytes

The RBCs struggle to pass through capillaries = Impaired oxygen transporting capacity

Question 18

what is familial hypercholesterolemia?

Answer 18

it’s a genetic disorder characterized by high cholesterol levels, especially of LDL (low-density lipoprotein) in the blood.

It shows the importance of Membranous Proteins e.g. LDL receptor.

Question 18

what are the reasons that lie behind the high levels of cholesterol?

Answer 18

• no receptors produced
• receptors never reach cell surface
• receptors can not bind LDL
• receptors do not internalise on binding
• receptros do not release LDL

Question 18

why are Biological Catalysts different from Chemical Catalysts?

Answer 18

[1] Catalyse high reaction rates

[2] Have a great reaction specificity

[3] Work in mild temperature/pH conditions

[4] Can be regulated

Question 19

what is a cofactor?

Answer 19

a non-protein chemical compound or metallic ion that is required for an enzyme’s activity as a catalyst, they can be seen as helper molecules.

Question 20

what are coenzymes?

Answer 20

Heat-stable substances that can aid enzyme reactions by loosely binding to their active site

e.g. FAD from riboflavin

Question 21

what are isoenzymes?

Answer 21

they are enzymes that catalyse the same reaction but vary in structure and other biochemical properties

Question 22

how do enzymes lower the activation energy?

Answer 22

1. Entropy reduction - Enzymes “force” the substrate(s) to be correctly orientated by binding them in the formation they need to be in for the reaction to proceed
2. Desolvation - Weak bonds between the substrate and enzyme essentially replace most or all of the H-bonds between substrate and aqueous solution
3. Induced fit - Conformational changes occur in the protein structure when the substrate binds

Question 23

what does the the Km and the Vmax tell us in the Michaelis-Menten Equation?

Answer 23

Km (Michaelis Constant) is the substrate concentration necessary to allow an enzyme to function at half its maximal velocity (1/2 Vmax)

It is also a measure of an enzyme’s affinity/specificity for its substrate under some circumstances:

Km tells us how much substrate the enzyme can take on:

A lower Km corresponds to a higher substrate affinity
(enzyme requires to take on smaller amount of substrate to reach Vmax)

A higher Km corresponds to a lower substrate affinity
(enzyme requires to take on larger amount of substrate to reach Vmax)

Vmax shows how fast a reaction is proceeding when the enzyme is saturated with substrate, it is dependant on enzyme concentration.

A lower Vmax corresponds to a slower enzyme

A higher Vmax corresponds to a faster enzyme.

Question 24

what is competitive inhibition?

Answer 24

it’s when an inhibitor binds to the active site of the enzyme and prevents the substrate from binding there.

In the presence of a Competitive inhibitor, it takes a higher substrate concentration to achieve the same velocity as no inhibition.

It can be overcome by adding more substrate.

Vmax is unaffected if there is enough substrate available.

To get to 1/2 Vmax more substrate is needed so the Km is larger.

Lineweaver-burk plot:

The Km increased (the red line on the X-axis)

The Km line went down the X-axis because the scale is 1/Km and dividing one by a bigger number will give a smaller number, that’s why although it is increased it is closer to zero not further.

The Vmax is unaffected (the interception line on the Y-axis).

Question 25

what is non commpetitive inhibition?

Answer 25

it’s when an inhibitor binds to a secondary site on the enzyme, changing the shape of the active site and preventing the substrate from binding, or it might be able to bind but the rate of product formation is reduced or stopped.

In the presence of a non-competitive inhibitor, there is a reduction in the number of functional enzyme molecules that can carry out a reaction so:

Vmax is reduced while Km remains the same.

That is because the active site of the enzymes that have not been inhibited is unchanged.

Question 26

Why is enzyme activity measured in a clinical setting?

Answer 26

Detection of suspected disease at pre-clinical stage

Confirmation of suspected disease and assessing severity

localisation of disease to organs

Characterisation of organ pathology

Assessing the response to therapy

Organ function assessment

Assessing genetic susceptibility to drug side effects

Detection of inherited metabolic disease

Detection of vitamin deficiencies

Question 27

What factors can influence enzyme activity in samples?

Answer 27

There are some enzymes that are secreted from cells, but generally serum enzyme concentrations are low and rise only when there is damage to cells and release of contents.

Question 28

what processes trigger the release of enzymes from cells?

Answer 28

Hypoxia – loss of oxygen supply due to occlusion, or inadequate oxygenation, or loss of oxygen carrying capacity

Cellular damage due to chemicals, drugs

Physical damage due to trauma, surgery, burns, radiation etc.

Immune disorders – anaphylaxis, autoimmune disease etc

Microbiological agents– bacteria, viruses, fungi, protozoa, helminths

Genetic defects – many, e.g. Duchenne’s Muscular Dystrophy

Nutritional disorders – protein-calorie, vitamin, mineral deficiency

Question 29

How do we analyse enzymes in the lab?

Answer 29

Rate of an enzyme-catalysed reaction is directly proportional to the amount of enzyme present

Progress of conversion of the substrate to product is monitored (Fixed-time & Continuous)

Spectrophotometry - measurement of an end product

Electrophoresis – to determine type of enzyme

Question 30

What are the problems with enzyme measurement?

Answer 30

Not specific i.e. can be found in more than one tissue in the body.

Particular requirements – Optimal temperature, pH etc.

Assays must be optimised

Enzyme assays are performed to serve two different purposes:

(i) To identify a special enzyme, to prove its presence or absence in a distinct specimen, like an organism or a tissue.

(ii) To determine the amount of the enzyme in the sample.

Question 31

what is alkaline phosphatase?

Answer 31

it’s a hydrolase class enzyme present in the body as an isoenzyme i.e. it catalyses the same reaction but varies in structure and chemical properties.

Catalyses the cleavage of orthophosphate from orthophosphoric monoesters under alkaline conditions (don’t need to remember this)

It can orginate from:

Bone & Liver & Kidney & Intestine & Placenta

Question 32

how can enzymes be used clinically?

Answer 32

• Detect Tissue damage

e.g. detecting the levels of Creatine Kinase which might indicate skeletal muscle damage

• Determine origin of affected tissue

e.g. determining the origin of raised levels of Alkaline phosphatase

• Notice diseases related to enzyme defects

e.g. low/high TPMT activity may be due to genotype mutation

e.g. Alpha galactosidase defect due to X-linked disorder which causes Fabry disease.

e.g. anaesthesia affected by prolonged apnoea due to AF phenotype which decreases the activity of BChe enzyme.

Question 33

what is allosteric regulation?

Answer 33

Allosteric regulation is any form of regulation where the regulatory molecule (an activator or inhibitor) binds to an enzyme in a region other than the active site

The place where the regulator binds is called the Allosteric site

Question 34

Enzymes can be regulated by other molecules, which either be:

Answer 34

1- Activators - molecules that increase the activity of an enzyme

2- Inhibitors - molecules that decrease the activity of an enzyme

Question 35

allosterically-regulated enzymes vs Covalently modified enzymes.

What’s the difference?

Answer 35

allosteric-regulated enzymes are enzymes that have their activities regulated by the binding of modulators or effectors, which can be either activators or inhibitors, at the site other than the active site.

Covalently modified enzymes are enzymes that have been modified through processes that involve forming or breaking covalent bonds, which usually occur post-translation.