enzymes

• Enzyme = biological catalyst

  catalyst = a substance that speeds up the rate of a reaction by lowering the activation energy without itself undergoing a permenant change. since enzymes aren't permenantly altered by the reactions they catalyse they can be used again many times over
• properties of enzymes:

  • enzymes are globular proteins, their function is dependent on their exact tertiary structure, if the structure is changed or (denatured), the activity is reduce/lost
  • the enzye catalyses a change in a substrate (chemical to be altered in reaction) to convert it into a product (altered chemical/outcome of reaction), only a small part of the enzyme (active site) is in contact with the substrate
• properties of enzymes:

  • active site = 3D crevice in enzyme structure formed by folding of protein chain. region of enzyme where substrate molecule binds and undergoes a chemical reaction to form a product. Shape of active site is complementary to shape of substrate: substrate fits exactly in the active site, denaturiation of enzyme protein loses shape of active site which results in lowering rate of reaction
  • enzymes are extremely specific, only one type of substrate molecule may be altered by each enzyme ie substrate that fits into active site
• properties of enzymes:

  • enzymes have very high turnover number = no. of substrate molecules turned into product/second => small amount of energy can affect large amount of substrate
  • enzymes may be intra-cellular or extra-cellular
• enzyme nomenclature:

  • substrate enzyme works on, reaction that is carried out to convert substrate to product, suffix -ase
  • there are exceptions to nomenclature rule e.g. trypsin, pepsin, catalase
• name of enzyme, substrate it works on, function of enzyme

  • DNA polymerase, DNA -> extends DNA polymer = DNA replication
  • RNA polymerase, RNA -> extends RNA polymer
  • Lactate dehydrogenase, Lactose -> removed hydrogens
  • ATP synthase - ADP and Pi ->Synthesises ATP
  • Pyruvate decarboxylase - pyruvate -> removes CO2
  • lipase - lipids - hydrolyse triglycerides
  • amylase - amylose - hydrolyse amylose
  • Ribulose bis phosphate (RuBP) carboxylase oxygenase - RuBp -> fixes CO2 in process of p/s
  • carbonic anhydrase -> CO2 & H2O <-> H2CO3 -> removes water from carbonic acid to form CO2 or vice versa
• exceptions to nomenclature rule e.g. trypsin, pepsin, catalase
• Intracellular Enzymes:

  • act within cells e.g.: catalase, 2H202 (hydrogen peroxide) --> 2h2O + O2
  • how could the activity of this enzyme be measured experimentally? by measuring volume of O2 evolved over time
• Extracelluler Enzymes:

  • made inside cells (on ribosomes), work outside cells
  • secreted out of cells using secretory vesicles formed from the golgi apparatus by process of exocytosis
  • mostly digestive enzymes e.g. amylase, amylose --> maltose
  • how could the activity of this enzyme be measured? rate of substrate used up: iodine test for starch, rate of product formed: benedicts test
• Mechanism of enzyme action:

  A) enzyme substrate complex (ESC)

  B) enzyme product complex

  
• mechanism of enzyme action:

  • substrate binds to enzyme at active site to make a temporary and short-lived enzyme-substrate complex (ES complex)
  • substrate is changed to product in active site
  • an enzyme-product complex is formed
  • products released by enzyme => enzyme can funciton again i.e allow another subsrate to bind to active site
• concept of activation energy

  • enzymes are biological catalysts that speed up reactions:
  • they work by reducing/lowering activation energy
  • this is the energy required to initiate a reaction/reach the transition

  key definitions:

  • metabolism = all the chemical reactions that occur in a living organism
  • anabolism = synthetic reactions: monomers -> polymers, requires energy (endothermic)
  • catabolism - breakdown reactions: polymers -> monomers, hydrolysis, digestion, releases energy (exothermic)
• models for binding of substrate to active site:

  1. lock and key hypothesis -> shape of active site is exactly complementary to shape of substrate
  2. induced fit -> induced fit indicates a change in shape of active site in response to substrate binding

  • substrate(s) makes a successful collision with the active site
  • enzyme active site (and/or substrate) changes shape to fit substrate => induced fit
  • substrate becomes bound to enzyme by bonds made with amino acid residues in active site
  • this binding weakens (puts a strain on) existing bonds in substrate(s)/leads to lowered activation energy
• factors affecting activity of enzymes:

  • temperatue
  • substrate concentration
  • enzyme concentration
  • pH
• 1. substrate concentration

  • rate of reaction increases as substrate concentration increases i.e. the substrate is rate limiting:
  • as substrate concentration increases, rate of successful collisions increases, rate of formation of ES complexes increases, rate of reaction increases
  • intil all active sites are occupied/saturated . subsequent substrate molecules must wait for an available active site
  • substrate is no longer rate limiting, that is, any further increase in tis concentration has no effect on the rate of reaction - Vmax
• 2. enzyme concentration

  • rate of reaction reaction increases as enzyme concentration increases i.e. the enzyme concentration is rate limiting
  • under physiological conditions the concentration of enzymes is much less than substrates the graph does not level off and the enzyme remains rate limiting. under experimental conditions however, enough enzyme could be added for it to be in excess
• 3. pH

  • a slight change in pH disrupts ionic and hydrogen bonds involved in the formation of the tertiary structure of the protein enzyme, so enzyme is denatures
  • structure of the active site is lost (no longer complementary) substrate no longer fits
  • rate of reaction goes down
  • extracelluler enzyme have a broader range of pH than intracelluler enzymes
• 4. temperature

  • as temp increases so does reaction rate:
  • temperature increases
  • kinetic energy increases
  • successful collissionns increase
  • number of ES complexes formed increases
  • rate of reaction increases
  • until optimum temp is reached
  • after the optimum temp:
  • as temp increases: vibration energy (kinetic energy of bonds within enzyme) h bonds involved in formation of 3D structure break i.e. enzyme is denatures
  • so structure of the active site is lost and is no longer complimentary to substrate
  • substrate no longer fits
  • ES complexes cannot form
  • rate of reaction decreases
• Temperature coefficient Q10

  • the temp coefficient for an enzyme catalysed reaction is the ratio between the rate of the process at two different temps. which are 10C apart
  • temp coefficient (Q10) = rate of reaction at tC/ Rate of reaction at (t-10) C
  • where t is any chosen temp
  • Q10 for most enzymes is 2 up to optimum temp
  • after optimum temp Q10 < 1
• 5. Enzyme inhibitors

  inhibitor = molecule which reduces rate of reaction of an enzyme
• competitive inhibitors:

  • very similar structure to substrate
  • so can fit into active site
  • (BUT to reaction occurs, enzymes are specific for their substrates)
  • active sites become occupied so substrates cannot enter and ES complex cannot be formed
  • so rate of reaction is reduced
  • substrate and inhibitor compete for active site
• competitive inhibitors (part 2)

  • most types types of competitive inhibition are reversable and depend on the concentration of substrate and concentration of inhibtor
  • (some competitive inhibitors bind irrerversibly and are called inactivators
  • effect of competitive inhibition can be reversed by increasing concentration of the substrate
  • the likelihood of collision of the enzyme with substrate or inhibitor is dependent on the numbers of each molecule present
• non-competitive inhibitors:

  • non-competitive inhibitors don't compete with the substrate for active site
  • they bind (tightly) elsewhere on the protein enzyme molecule i.e. an allosteric site
  • an change the overall shape of the molecule
  • changing shape of an enzyme protein is an allosteric effect
  • so the shape of the active site is changed
  • and is no longer complementary to the substrate
  • substrate cannot enter active site
  • no products are formed
  • rate of reaction reduced
  • effect of non-competitive is non-reversible: cannot be reversed by increasing substrate concentration
• allosteric site:

  • a site other than active site:
  • when a molecule binds to an allosteric site, it changes shape of protein => also changes shape of active site
• End-product inhibition:

  • all enzymes catalyse reactions as part of a metabolic pathway
  • metabolic pathways must be regulated
  • a metabolic pathway includes a series of reactions from initital substrate to end product
  • initial substrate -- E1---> P1/S2 --- E2 ---> P2/S3 -- E3 ---> end product
  • end product inhibition of 1st enzyme pathway (negative feedbak)
  • S = Substrate, P = Product, E = Enzyme
• end-product inhibition:

  • if the pathway continues unregulated the end-product will be surplus to cell's requirements (and energy wasted in its making)
  • in most pathways, the end product is a reversable non-competitive inhibitor first enzyme of the pathway
  • the whole pathway stops and intermediates and energy are not wasted unecessarily
  • if pathway stops: concentration of end product gors down, enzyme inhibition is reduced, metabolic pathway starts again
  • this poress is an example of negative feedback and is called end-product inhibition
• enzyme cofactors:

  (umbrella term for all non-protein molecules which help enzymes work)

  enzymes may require a cofactor or co-enzyme to work. this is a molecule NOT made of protein