Factors that effect enzyme activity

What can the rate of an enzyme catalysed reaction be affected by?

• Enzyme concentration

• Substrate concentration

• Temperature

• pH

• Presence of inhibitors

Measuring enzyme controlled reactions- initial rate of reaction

Rate= formation of a product in a given time

• Reactions are always fastest at the start

• When the enzyme and substrate are first mixed there is greater chance of collisions with the active site

• As reaction proceeds, substrate molecules have been used up and less are available to the active sites

• As a result, the frequency os collisions decreases so the rate of reaction gives the maximum reaction rate for an enzyme

Effect of enzyme concentration

• As enzyme concentration increases, so does initial rate of reaction

• So long as there is sufficiently excessive substrate then the rate of a reaction will increase proportionally to the enzyme concentration

• This is because there are more enzymes for the substrate to collide with, forming more ESC’s and catalysing more reactions

Effect on enzyme concentration graph

• Rate plateaus is sub conc is fixed- sub conc is the limiting factor

• Linear relationship- directly proportional- as one increases, so does the other- enzyme conc is limiting factor

• As enz conc increases, more available active sites to catalyse reaction, so rate of reaction increases

Effect of enzyme concentration on living cells

• In living cells, the availability of enzymes depends on the the rate of synthesis/ degradation

• Genes for synthesising enzymes can be turned on/off depending on the cells needs

• Old enzymes are degraded by the cell into amino acid and used to synthesise new enzymes (enzymes don’t age- refers to recycling molecules)

• This also removes proteins which are abnormally shaped and regulates metabolism of the cell

Effect of substrate concentration

Limiting factor- any factor that is determining how quickly a reaction is taking place

• Initial rate os measured over 30p seconds for different substrate concentrations

• The rate rises to a maximum (A) and then slows and levels off: plateaus (B) Because all active sites are occupied so rate does not increase

• A- rate increases as substrate concentration increase so substrate is limiting factor

• B- enzyme conc becomes limiting as all active sites are filled (known as V max), rate would increase again if there were more available enzymes

Effect of temperature

• At low temperatures, increase temperature increases rate of facet ion due to increase kinetic energy of molecules:

  • Low energy- low collisions- not enough Ek

  • Heating up- more successful collisions with the required activation energy

• However, at high temps a decrease in activity observed due to thermal desaturation of enzymes

Temperature: optimums

• Our mammalian enzymes work at 37 degrees c (not all enzymes)

• Range is specific to organisms and where they live

• For some microbial enzymes, particularly those in thermophilic bacteria, optimal temp is as high as 65 degrees c

• Temp is often given as a range as pinpointing the exact optimum is challenging

Temperature: link to proteins

• Enzymes are globular proteins whose structure and active sites is determined by bonding between R groups

• High temps = H-bonds vibrate more in the molecule itself between R groups, causing them to break, the active sites shape changes so the substrate no longer fits, and a reaction doesn’t occur (tertiary structure is disrupted)

Q10- the temperature coefficient

• It is the increase in rate when temp is increased by 10 degrees c

• When Q10 is exactly 2, rate has doubled, 3= tripled rate

• Usually calculated from a graph

Effect of pH

• There is an output of pH for each enzyme catalysed reaction

• Works fastest at optimal pH- concentration of hydrogen ions determines pH

• More ions= lower pH

• Differences of x10 doer each pH, e.g. pH 1 to 3= 100 ions

• At other pH values, enzymes may function less efficiently to not at all

• Pepsin- found in stomach

• Chymotrypsin- found in small intestine

• Changes in pH can break ionic or hydrogen bonds between R groups, which may result in a shape change of active site, so substrate no longer fits= denatured

• Small changes in pH can be reversed as the active site is disrupted slowing the reaction, but its not permanently changed

• Large changes of pH will be irreversible

pH and location

• Extracellular enzymes may have an optimum pH different to pH 7 depending on location

• Salivary amylase- pH 6-8

• Pepsin in the stomach- pH 1-2

• Trypsin and enterokinase digesting protein in the small intestine work in pH 7-8 (more alkaline than stomach as bile salts are added

Effect of inhibitors

Inhibitors, when added to an enzyme/ substrate mixture, reduce the rate of reaction

There are two types of reversible inhibitor: competitive and non-competitive

Reversible inhibitors: when removed, active sites returns to normal shape

Non-reversible inhibitors: change shape of active sites permanently

Competitive inhibitors

• A competitive inhibitor has the same shape as the substrata molecule

• They compete for the same active site

• If the inhibitor enters the active site first, it means the substrate wont be able to bind

• The relative concentration of inhibitor and substrate will determine the number of active sites occupied by inhibitors and the degree of inhibition

• The effect on rate of reaction of higher the more inhibitors

• The higher the concentration of substrate, the less inhibition occurs

• Inhibition of competitive inhibitors reduces rate of enzyme activity

Non- competitive inhibitors

• The enzyme has two sites, active site for substrate and allosteric site where the inhibitor may bind

• If no inhibitor, the substrate can bind to the active site but the inhibitors can bind to the allosteric site

• This causes a change in their 3D shape of their active site pf the enzyme, meaning the substrate can no longer fit (allosteric hindrance)

• The amount of substrate and inhibitor determine the rate of reaction

End product inhibition

• After a catalysed reaction has completed, product molecules may stay bound to the enzyme, preventing thre enzyme making more of this product

• Negative feedback

• Example of a metabolic pathway:

  • Substrate A converted to B, B-C, C-D, D-E

  • End product E can bind to to enzyme 1 and acts as a non-competitive inhibitor, this means that end product E wont build up in the cell, because E is made, it slows down the formation of itself by inhibiting the first enzyme in the sequence

Examples

• Enzymes and poisons- cyanide

• Enzymes in medicine

• Aspirin

• Antibiotic resistance

• Snake venom

Enzymes and poisons- cyanide

• Some poisons work by blocking the action of enzymes

• Cyanide, block the action of cytochrome oxidase- an enzyme involved in the final stages of respiration in the mitochondria, cyanide binds H+ there which stops aerobic respiration

• It is a non-competitive inhibitor

• Only 100-200 mg can make you lose consciousness, this can happen in under 10 seconds

Enzymes in medicine

• Can be used to combat infections caused by viruses

• E.g. protease inhibitors stop replication of HIV in its tracks as it stops virus being able to make its protective protein coat

• This is a competitive inhibitor

• Revers transcriptase inhibitors block this enzyme which is essential for viral replication

Aspirin

• Combine to enzymes that synthesise prostaglandins- molecules that are important for detecting and repointing to pain, they’re signalling molecules

• Cyclooxygenases (COX enzymes) are also responsible for their synthesis and are the sites for other drug use

Antibiotic resistance

• Antibiotics are out first, sometimes only, line of bacterial defence

• Bacteria have evolved an enzymes that can break down antibiotics such as penicillin- beta lactamase

• These bacteria alter then said to be resistant to any bacteria that has a beta lactam ring

Snake venom

• Lethal dose of venom and enzyme

• Phosphodiesterases are also present in snake venom, affecting functions of the victims hear- some also affect DNA backbones

• Also contain acetyl cholinesterase- blocks nerve transmission

• Also contain hyaluriclurase- digests connective tissue