Enzymes

Enzyme - protein molecule that acts as a biological catalyst. 

Enzymes are globular proteins, with a specific teriary structure, which catalyse metabolic reactions in living organisms. (a)

Processes that require enzymes: protein synthesis; digestion; respiration; and protein sythesis. Need enzymes to break down gylcosidic, ester and peptide bonds.

Similarities 

• generally soluble
• specific - one enzyme per substrate often
• globular structure contains a cleft area - active site
• activity affected by by temperature and pH.

Active Site

Active site is a tiny part of enzyme - often <10 out of hundreds of amino acids.

Activation Energy - amount of energy that must be applied for a reaction to proceed. Different reactions require different levels of activation energy. Enzymes reduce the amount of activation energy needed to allow a reaction to take place.

Biologically important molecules are too stable to break up or self assemble under conditions found in the cell. As cells are unlikely to survive methods used in labs (boiling, acid etc) our bodies use enzymes to drive metabolic reactions. 

Enzymes work by reducing the amount of activation energy required because of the way the active site is shaped to fit the substrate molecule. 

Lock and Key Hypothesis

Enzymes have specifically shaped active sites, which are complementary to the shape of the substrate molecules involved in the reaction. In this hypothesis the substrate 'key' fits into the active site 'lock', which keeps the substrate held in one place to allow the reaction to take place. 

Induced Fit Hypothesis

This states that the active site isn't exactly complementary to the substrate. Instead the enzyme molecule slightly changes its shape when it collides with a substrate. Making the substrate fit into place because oppositely charged groups on the substrate and active site are found near each other. The change in enzyme shape destabilises the substrate molecule so the reaction can occur more easily. 

Extracellular - enzymes catalyse reactions outside the cell.

Many enzymes in (an internal) digestive system are released from cells that make them onto food in digestive system spaces. Whilst some organisms secrete enzymes onto their food outside themselves then organism takes in the monomers.

Intracellular - enzymes catalyse reactions inside the cell.

These can be found in the cytoplasm of cells or attacted to cell membranes, like lysosomes.

(b)

Endotherms

Endothermic animals can maintain their internal body temperatures which means enzymes can function at near optimum temperatures at all times. This requires a lot of energy to maintain it however it makes endotherms able to survive in a variety of environments. 

Protection

Animals use enzymes as a defense mechanism too. Phagocytes take in and digest bacteria using hydrolytic enzymes in the lysosomes to destroy invading microbes like bacteria. 

Denaturation - changes the enzymes tertiary structure such that it cannot function and its function cannot be restored. Doesn't change primary structure of protein.

Applying heat to an enzyme gives it more kinetic energy and makes it vibrate.

These vibrations can break the weaker bonds (hydrogen and ionic) which are responsible for maintaining the shape of the tertiary structure.

The more you heat an enzyme the more bonds are broken, which slowly makes the tertiary structure lose it's shape. This decreases rate of reaction.

Eventually enough bonds will break for the tertiary structure to unravel and the enzyme will permanently stop working. 

(d)

Optimum Temperature - temperature which gives fastest rate of reaction in enzyme controlled reactions.

Increasing temperature increases rate of reaction initially but eventually the rate will begin to decrease until the enzyme stops working.

O.T is a balance between increasing kinetic energy (thus inc. no. of collisions) and increasing vibration of the enzyme molecule.

O.T varies depending on the organism's environment and internal temperature. Most have their O.T between 40 and 50 degrees C.

(d)

Measuring effect of temp. on enzymes.

Carry out enzyme controlled reaction at different temperatures, using a thermostat controlled waterbath. Measure production of product or the disapearance of the substrate.

(e)

Optimum pH - pH value at which the rate of an enzyme controlled reaction is at its maximum. Each enzyme has a specific optimum pH.

Only extreme changes (three values etc) in pH denature enzymes, (not minor changes) distrupted bonds can reform if pH goes back to optimum.

Most enzymes O.pH is around 7, where the concentration of hydrogen ions gives the tertiary structure the best overall shape and holds the shape that is complementary to the substrate.

Different organs in organisms can have very different pHs, e.g. stomach and small intestine.

The higher the concentration of H+ the lower the pH value. This positive charge is attracted to negative charges and repelled by like charges in tertiary structure. Hydrogen ions can interfere with hydrogen and ionic bonds which alters the tertiary structure thus the active site.

Increasing conc. of hydrogen ions alters the charges around active sites as they tend to 'cluster' around negatively charged groups/ions. Thus decreasing rate of reaction.

(d)

Measuring effect of pH on enzymes

Carry out enzyme-controlled reactions at different pH values using buffer solutions aand measure the production of a product or disappearance of a substrate. (e)

IN EXPERIMENTAL CONDITIONS.

Enzymes

Conc. of enzyme is varied for a fixed concentration of substrate molecules.

As enzyme conc. increases more active sites become available, thus more enzyme-substrate complexes form, thus inc. rate of reaction.

Will reach a maximum rate in which all substrate molecules are occupying all enzyme active sites.

Substrate

Conc. of substrate varied for fixed conc. of enzyme molecules.

As substrate conc. increases more enz-sub complexes form because of increase of collisions. (Again) inc rate of reaction.

Will reach max rate of reaction.(e)

Initial Reaction Rate - highest reaction rate which gives max. possible rate of reaction for an enzyme under experimental conditions.

As reaction proceeds, product molecules are formed and increase in number, whilse substrate molecules are used up and decrease in number. Therefore rate of reaction decreases as reaction continues. 

Limiting Factors - if all other conditions were kept constant, increasing the concentration of that factor alone would increase rate of reaction.

Cells

Conc. of enzymes kept at a constant low level because enzymes can work over and over again, driving the same reaction. To regulate enzyme activity, cells adjust concentration of enzymes and/or substrates. 

(d)

Enzyme Inhibitor - any substance or molecule that slows down the rate of an enzyme-controlled reaction by affecting the enzyme - temporarily or permanently - in some way.

Competitive inhibitors have a similar shape to substrate molecules, allowing them to occupy the active site and forming enzyme-inhibitor complexes.

• Reducing the number of enzyme-substrate complexes that can form.
• Preventing the enzyme from catalysing a reaction.
• Reduces rate of reaction.

Level of inhibition depends on concentration of inhibitor and substrate, the more inhibitor molecules present the more inhibitors collide with active sites thus the effect of inhibition is greater (and vise versa).

(f)

N-C inhibitors bind to the enzyme molecule in a region away from the active site - an allosteric site. This bonding distorts the tertiary structure of the enzyme thus the shape of the tertiary structure.

The substrate no longer fits into the active site to form enzyme-substrate complexes. Changing the concentration of the substrate will have no effect on this type of inhibition.

Many N-C inhibitors are permanent and non reversible, they effectively denature the enzyme. Whilst most competitive enzymes only bind for a short period, leaving the enzyme molecules unaffected.

Organisms use inhibitors of both kinds to control the rate of reaction in enzyme-controlled reactions like respiration and photosynthesis.

(f)

Cofactor is any substance that must be present to ensure enzyme controlled reactions take place at the appropriate rate. Some are part of the enzyme and others affect it on a temporary basis.

Coenzymes e.g. Vitamin B3 (nicotinamide)

• Small organic, non protein molecules that bind to the active site for a short period - either just before or at the same time as the substrate bonds. 
• Take part in the reaction and are changed in some way. Unlike substrates they are recycled back into the reaction. 
• Role: carry chemical groups between enzymes so they link together in enzyme reactions that need to take place in a specific order. 

Prosthetic Group e.g. enzyme carbonic anhydrase contains zinc prosthetic group.

• permanent part of the enzyme.
• Vital to function and tertiary structure of enzyme. 

Inorganic Ion Cofactors. 

• Can increase rate of reaction
• Binding of ion makes enzyme-substrate complex form more easily because it affacts the charge distibution amoung other things. 
• Example: amylase will only function properly if chloride ions are present. 

Biosenser - uses enzyme controlled reactions to detect the presence of substances in a highly sensitive and specific way.

Enzymes are used as part of the treatment for cyctic fibrosis. A symptom of CF is the passage of digestive enzymes from the enzyme to gut, thus meaning that individuals with this condition find it difficult to digest food. Doctors prescribe enzymes that are in an acid resistant coat to prevent erosion from the stomach acid and come in the form of pills.

Viral infection llike HIV are also treated using chemicals that act as viral protease inhibitors. The inhibitors prevent the virus from being able to build new virus coats.

Penicillin is an inhibitor of a baterial enzyme that forms cross links in the cell wall of some bacteria, which prevents bacterial reproduction. Many strains of penicillin are resistant to penicillin because they produce beta-lactamase which breaks down the penicillin molecule.

The same enzyme inhibitor can be both a poison and drug depending on dosage and location of inhibitor.

(i)

Metabolic poisons are often enzyme inhibitors, they are also capable of overactivating enzymes. 

Ethylene Glycol, an ingredient in engine antifreeze, itself isn't poisonous but if taken in by the body is broken down in the liver by dehydrogenase producing oxalic acid which is extremely toxic. To overcome this, a patient will be given a massive dose of(ethanol) alcohol which acts as a competitive inhibitor of alcohol hydrogenase reducing rate of production of oxalic acid. 

Snake Venom is a mixture of toxins and enzymes, phosphodiesterase causes a fall in blood pressure; acetyl cholinesterase - paralysis; hyaluronidase - break down connective tissues; APT-ases disrupt the preys use of energy.

Cyanide is a non competitive inhibitor for a vital respiratory enzyme called cytochrome oxidase, which can be found in the mitochondria. It prevents the production of ATP forcing the body to respire anaerobically which leads to a build up of lactic acid. Can kill an adult in 2 hours.

(h)

Turnover Number - of an anzymeis the number of reactions an enzyme can catalyse in one second.

In most enzyme controlled reactions, reactions are limited (in rate) by how fast the substrate can collide with te active site. This means we must control these reactions to prevent damaging our cells. 

Respiration and photosynthesis are examples of complex metabolic sequences, meaning the product of one enzyme controlled reaction is the substrate in the next enzyme controlled reaction. This chain is called a metabolic pathway. 

To prevent a build up of an unwanted product, the end product can attach to one of the enzymes early in the sequence - acting as a reversible non-cmpetitive inhibition. The end product binds to the enzyme away from the active site, changing the shape of the active site and reducing the rate of reaction. 

Essential Enzymes...

Some enzymes found in all organisms appear to be vital for life itself. E.g. ATP Synthase catalyses the addition of an inorganic phosphate group to an ADP molecule. ATP synthase can be found in animals, bacteria and plants. 

Inborn Errors of metabolism

If the DNA forming the instructions for an enzyme has mutated then the enzyme may not be made correctly which is the foundation for many diseases (the lack of a functioning specific enzyme in a metabolic sequence), e.g. phenylalanine.