Lecture 12 - Enzymes I Flashcards
Enzymes (1)
Proteins that speed up (catalyse) specific chemical reactions.
Properties (5)
- Increases reaction rate (by up to 10 billion-fold)
- Show specificity
- Unchanged at end of reaction
- Do not alter reaction equilibrium
- Facilitate reaction by decreasing the free energy of activation of the reaction
Functions (5)
- Digestion: carbohydrates, fats, proteins
- Blood clotting: fibrin clot catalysed by thrombin
- Defence-immune system-activation of complement
- Movement: muscle actomyosin is an ATPase
- Nerve conduction: membrane ion pumps for Na+, Ca+2
Intracellular/Secreted(3)
- Catalyse a particular type of reaction.
- Proteases (uses water e.g. hydrolysis), nucleases, polymerases, kinases, etc.
- Enzyme defects cause disease.
Intracellular/Secreted - Diseases (6)
• Phenylketonuria - Cannot convert Phenylalanine to Tyrosine. Cannot make melanin, and the accumulation of phenylalanine in their blood leads to intellectual disability, seizures, behavioural problems and mental disorders.
New born babies are screened for PKU with a heel prick test. So that if the result is positive their diet can be adjusted to include very little phenylalanine, to prevent irreversible brain damage.
• Glycogen storage disease – Mutations in enzymes that mobilise glucose from glycogen in the liver.
Livers main function is to buffer glucose in the blood, defect = glucose cannot be regulated properly in the blood.
• Tay-Sachs disease - not making enough membrane cerebroside.
Absence of a vital enzyme called hexosaminidase-A (Hex-A).
Without Hex-A, a fatty substance, or lipid, called GM2 ganglioside accumulates abnormally in cells = progressive damage to the cells.
Drug targets (6)
- Antibiotics- penicillins inhibit cell wall synthesis.
- Anti-inflammatory agents- aspirin block prostaglandin. Aspirin blocks a prostaglandin enzyme; therefore, it reduces effects of swelling/oedema.
- Anticancer drugs- methotrexate is a folate analogue: interferes with synthesis of DNA precursors, targets enzymes involved with DNA replications.
Active sites + Evidence (1 + 2)
• 3-D cavity or cleft that binds substrate(s) using electrostatic, hydrophobic, hydrogen bonding and van der Waals interactions.
• Evidence from:
o X-ray crystallography as you can see where the enzyme and substrate binds, can identify the key residues for binding.
o Kinetic studies of enzyme activity.
Active site - Example (5)
RAS protein it is a switch found on the inside of plasma membrane, switched on with GTP bound to it.
Hydrolyses it to GDP and P and becomes inactivated.
In tumour cells the RAS protein is mutated in 50% of human cancers, the effect of the mutation is that it deactivates GTPase.
The RAS Protein is turned on and communicates to all other proteins in the cell and tells them to grow. Leading to the formation of tumours.
Lock and key hypothesis (4)
- The specific indented area on the surface of the enzyme molecule (active site), is where the substrate molecules fit. They fit because the tertiary structure of the enzymes/ active site gives it a shape that is complementary to that of the substrate molecule.
- Substrate binds to active site forming an enzyme-substrate complex.
- The substrate then reacts and forms an enzyme-product complex.
- The product is then released leaving the enzyme unchanged.
Induced fit hypothesis (4)
- When the substrate molecules fit into the enzymes active site it changes shape slightly to mould itself around the substrate molecule.
- The active site still has a shape complementary to the shape of the substrate molecule. On binding, the subtle changes of the shape of the side chains (R-groups) of the amino acids that make up the active site give a more precise conformation that exactly fits the substrate molecule.
- An ESC is formed and non-covalent forces such as hydrogen bonds, ionic attraction, VdW forces and hydrophobic interactions, bind the substrate molecule to the enzyme’s active site.
- E+S ESC EPC E+P
Induced fit hypothesis - Example (3)
Hexokinase uses glucose and ATP as it substrates, if you leave out ATP, you can crystallise the enzyme alone with glucose forming ESC, and then the two lobes of the hexokinase structure come together as glucose binds to the active site, enzyme changes shape, conformational change.
Factors responsible for enzyme catalysis - ES binding energy is used … (6)
o To bring molecules together in active site- A + B = C + D.
o To constrain substrate movement.
o Stabilise positive and negative charges in t-state.
o Provide a reaction pathway of lower energy- e.g. involving covalent enzyme-substrate intermediates.
o To strain particular bonds in the substrate- making breakage easier. Substrate distorted on binding to resemble transition state. e.g. lysoszyme
o Use cofactors: bring new chemistry to active site.
Competitive inhibition (10)
Inhibitor that competes with substrate to bind to active site on an enzyme.
Similar shape and it blocks the active site and prevents the formation of an ESC.
The amount of inhibition depends on the concentration of substrate and inhibitor molecules.
More inhibitor = greater inhibition -= reduces rate of formation of ESC
More substrate = less inhibition.
Most competitive inhibitors bind reversibly to the active site.
Binds irreversibly = inactivator.
Reduced ROR.
Vmax is unaltered.
Km is increased. I.e. the reaction can still reach its full velocity, but to get to half of that maximum velocity, you need much more substrate to outcompete the inhibitor.
Competitive inhibition - Examples (4)
Statins, used in the synthesis of cholesterol. Regularly described to help people reduces blood cholesterol concentration. High blood cholesterol level can result in heart disease.
Aspirins irreversibly inhibits the active site of the COX enzymes, preventing the synthesis of prostaglandins and thromboxane (chemicals responsible for pain and fever).
Non-competitve inhibition (7)
Binds to an enzyme at an allosteric site.
Attaches to allosteric site, which disrupts enzyme tertiary structure and change its shape so an ESC no longer forms and it is no longer complementary.
Maximum ROR is reduced, adding more substrate will not really affect this.
Vmax is decreased.
Km remains unaltered i.e. there are less active sites available, but the affinity of the active sites hasn’t changed as the inhibitor is binding to another site.
More inhibitor = greater inhibition -= reduces rate of formation of ESC.
Can be reversible or irreversible.
