Enzymes 1 Flashcards
Define ‘enzyme’
Proteins that speed up (catalyse) specific chemical reactions.
Describe diseases associated with enzymes
Phenylketonuria - Cannot convert Phenylalanine to Tyrosine. As a result, sufferers cannot make melanin, and the accumulation of phenylalanine in their blood leads to intellectual disability, seizures, behavioural problems and mental disorders. As this enzyme deficiency is so severe, all 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. Amino acid defect.
Glycogen storage disease - come about from mutations in enzymes that mobilise glucose from glycogen in the liver. Livers main function is to buffer glucose in the blood. If there is a defect in these enzymes, then glucose cannot be regulated properly in the blood. Sugar defect.
Tay-Sachs disease - membrane cerebroside. Not being able to make enough cerbroside (any of a group of complex lipids present in the sheaths of nerve fibres).
Tay-Sachs is caused by the 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, especially in the nerve cells of the brain. This ongoing accumulation causes progressive damage to the cells. Membrane defect.
Describe 3 drugs that target enzymes and the transition state graph
• Antibiotics: e.g. penicillin’s inhibit cell wall synthesis
• Anti-inflammatory agents: aspirin block prostaglandin
• Anticancer drugs: methotrexate is a folate analogue: interferes with synthesis of DNA precursors
Here we release free energy and the reaction is favourable. The reaction goes under the dotted line, the transition state is where the reaction can either fall back to the substrate or proceed to the product. You must put energy in to proceed to a product.
Describe active sites and RAS protein
3-D cavity or cleft that binds substrate(s) using electrostatic, hydrophobic, hydrogen bonding and van der Waals interactions
• Evidence from: X-ray crystallography and Kinetic studies of enzyme activity
RAS protein it is a switch found on the inside of plasma membrane, switched on with GTP bound to it, 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.
Describe factors responsible for enzyme catalysis
• Enzyme-substrate binding energy is used:
To bring molecules together in active site- A + B = C + D
To constrain substrate movement
Stabilise positive and negative charges in t-state
Provide a reaction pathway of lower energy: e.g. involving covalent enzyme-substrate intermediates
To strain particular bonds in the substrate- making breakage easier. Substrate distorted on binding to resemble transition state. E.g lysoszyme (muramidase or N-acetylmuramide glycanhydrolase is an antimicrobial enzyme produced by animals that forms part of the innate immune system- found in tears, break down the cell walls of pathogenic bacteria).
Use cofactors: bring new chemistry to active site
Describe the velocity - concentration graph
• The reaction velocity is the rate at which the products are made from the substrate. The Vmax is the maximum possible velocity when all active sites are occupied, the Vmax2 is half the max velocity when half of the active sites are occupied. The curve shows saturation behavior as it increases steeply but then begins to level off and plateau (shows that active sites is limiting factor, as no increase in rate, hence evidence for active sites) as there are less active sites available. Km is the amount of substrate you would have to put in to half saturate the active site (tells you about the binding affinity of the enzyme).
Describe the 2 equations
- First equation explains the curve above, second gives you a straight curve which can be used to determine the various factors, which can be seen above. It is a way of linearizing the data.
- The Km values of enzymes vary quite significantly
Describe competitive inhibition
- An inhibitor that competes with substrate to bind to active site on an enzyme.
- Have a similar shape to that of the substrate molecule and competes with the substrate for the enzyme’s active site. 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.
- If it binds irreversibly it is called an inactivator.
- Reduced ROR but does not change the Vmax of the enzyme it inhibits.
- The Vmax is unaltered, but the 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
- e.g. 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.
- e.g. Aspirins irreversibly inhibits the active site of the COX enzymes, preventing the synthesis of prostaglandins and thromboxane (chemicals responsible for pain and fever).
Describe non-competitive inhibition
- An inhibitor that 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 by presence of NCI. Adding more substrate will not really affect this.
- The Vmax is decreased, but the 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.
- e.g. Organophosphates used as insecticides and herbicides irreversibly inhibit the enzyme acetyl cholinesterase (necessary for never impulse transmission). Can lead to muscle cramps, paralysis and even death if ingested.
- e.g. Proton pump inhibitions (PPIs) are used to treat long term indigestion. Irreversibly block an enzyme system which is responsible for secreting hydrogen ions into the stomach. Makes PPIs very effective in reducing the proportion of excess acid which if left untreated can lead to the formation of stomach ulcers.
Describe the mechanisms that regulate enzyme activity
- Control of gene expression-enzyme amount
- Compartmentation: sequences in enzyme polypeptide chain target enzyme to ER, mitochondrion, nucleus etc
- Allosteric regulation: regulatory molecules control protein shape-increase (or decrease activity)
- Covalent modification of enzyme.
- Change enzyme shape and activity- e.g. phosphorylation
Describe feedback inhibition
In a metabolic reaction, substance A is converted through a number of steps to Z. If we want to regulate this pathway, we can do this through feedback inhibition. Which is as the product builds up, this inhibits the reaction, of A. So, this is done by allosteric regulation. The more of a product made the more it inhibits its production. e.g. glycolysis, cascade of enzymes used
Describe ATcase - C6R6
- ATCase-regulated by CTP (used in making RNA).
- Aspartate carbamoyltransferase catalyses the first step in the pyrimidine biosynthetic pathway. In E. coli, the enzyme is a multi-subunit protein complex composed of 12 subunits. The composition of the subunits is C₆R₆, forming 2 trimers of catalytic subunits and 3 dimers of regulatory subunits.
- Feedback inhibition is caused by CTP binding to ATCase.
- X Ray crystallography shows that the ATCase spindles holds two parts together.
