week 6 - enzymes

• Enzymes

  • theyre biological catalysts
  • they increase the rate of biological reactions
  • MOST enzymes are proteins
  • enzymes are SPECIFIC for their given reaction.
• why do we need enzymes

  • enzymes increase the rate of biochemical reactions (catalyse)
  • many enzymes are part of coordinated pathways, catalysing stepwise reactions
  • they are very specific for their substrate
• cool fact

  • many diseases are due to enzymes not functioning properly
  • many drugs target enzymes
• enzymes more facts

  • they are NOT used up during the process
  • enzymes are mostly GLOBULAR proteins
  • the native structure is critical for the activity of enzyme
• enzyme examples

  • DNA polymerase: enzyme related to DNA replication
  • Hexokinase: in glycolysis, involving phosphorylation of glucose to glucose 6-phosphate
  • Pyruvate dehyrogenase (PDH): pyruvate --> acetal-CoA important metabolism reaction

  A) DNA polymerase

  B) hexokinase

  C) pyruvate

  
• active site

  • the region in the enzyme responsible for catalysing whatever reaction
  • it is where the substrate binds to, for catalysis
• the process

  1. enzyme + substrate = enzyme-substrate complex (ES)
  2. it becomes the enzyme-product (EP) complex
  3. then it's Enzyme + product (E+P) because enzyme is unchanged

  
• reaction coordinate graphs
• reaction coordinate graph

  A) activation energy

  
• how to speed up reaction

  • increase temperature
  • lower the activation energy (enzymes)
  • change the pathway of reaction (enzymes)
• uncatalysed vs enzyme-catalysed reaction

  A) lower

  B) reaction coordinate

  C) free

  
• example

  1. without enzymes, bending a metal stick requires lots of energy
  2. at the point right before it bends, it can return back to straight or be bent
  3. this is called transition state (green)

  A) transition state

  
• disadvantage of lock and key model

  • the enzyme SLOWS the rate of reaction (bc it's locked)
  • since it requires more free energy (G) to reach transition state, than uncatalysed

  
• induced fit model

  • shows how the enzyme's active site adjusts to that of the substrate
  • results in lower activation energy

  
• induced fit model steps

  1. enzyme binds to substrate weakly but enough to be specific (forms ES complex)
  2. enzyme undergoes conformational change to bind tighter to the transition state (lower activation energy)
  3. ES complex is converted to enzyme-product
  4. Enzyme releases the product

  
• common amino acids as enzymes

  • tend to have side acidic and basic forms of side chains
  • Eg. Histidine's side chain has a pKa of around 7, meaning it can easily exist as both protonated and deprotonated form

  
• terms

  • cofactor: one or more INORGANIC ions involved in the active site reaction (eg. Fe2+, Mg2+, Zn3+)
  • coenzyme: a larger ORGANIC molecule that carries electrons or functional groups required for catalysis, mostly vitamins.
  • prosthetic group: a coenzyme or metal ion covalently tightly bound to the enzyme. (eg. heme in hemoglobin)
• quantifying enzyme kinetics

  • dependent on how much product is made (DV)
  • in the graph, it plateaus because the substrate eventually runs out
  • the dotted lines = initial velocity (V0), shows rate

  A) product

  B) time

  C) initial velocity

  
• graphing initial velocity against substrate concentration

  • as [S] increases, V0 reaches an asymptote at Vmax (initial velocity is maxed)
  • Km is the [S] that is half of Vmax
  • this graph is used to determine an enzyme's optimal perfomance
• graphing initial velocity against substrate concentration

  • as [S] increases, V0 reaches an asymptote at Vmax (initial velocity is maxed)
  • Km is the [S] that is half of Vmax
  • this graph is used to determine an enzyme's optimal perfomance

  A) Km

  B) substrate

  C) initial velocity

  D) Vmax

  
• Michaelis constant (Km)

  • the substrate concentration at which V0 is one-half Vmax.
  • it is for a GIVEN enzyme concentration
  • Km is proportional to enzyme concentration
  • Km NEVER changes
• lineweaver-Burk plot - 1/V0 AGAINST 1/[S]

  • this is MORE USEFUL
  • gives a straight line
  • 1/y-int = Vmax
  • -1/x-int = Km
  • Km/Vmax = slope

  A) slope

  
• Kcat constant

  • the maximal rate for an enzyme at saturation with substrate
  • it represents the NUMBER of substrate molecules CONVERTED into product in a given unit of time.
  • aka. enzyme's turnover rate.
  • It has units of "per time". 
• enzyme's are saturated when..

  • they are working at their maximum
  • due to the maximum amount of substrate
• do these values change or not

  • V0: changes with [S], but plateaus near Vmax
  • Vmax: proportional to [E]
  • Km: constant
  • Kcat: constant (?)
• question

  • Vmax=260 and Km=1 AT[E]=100nM
  • Vmax=? and Km=? AT [E]=200nm

  A) 520

  B) 1

  
• Km trends

  • the lower the Km, the more efficient
  • lower Km = enzyme requires less substrate to achieve half Vmax
• enzyme INHIBITION

  • the action of enzymes can be inhibited (reduced)
  • types: IRREVERSIBLE and REVERSIBLE
  • types: competitive vs uncompetitive
• inhibition types

  • irreversible: when a molecule permanently bonds to an enzyme, forming covalent bond. resulting in non-reactive enzyme
  • reversible: when a molecule binds and unbinds from enzyme. Enzyme action comes back after inhibitor is removed.
• competitive inhibition

  • comp: the inhibitor COMPETES with the substrate for binding to enzyme
  • diagram: 2 possible pathways

  A) competitive

  B) active site

  
• uncompetitive inhibition:

  • inhibitor binds to a SEPARATE site on the ES complex
  • the conformational change (E+S) allows inhibitor to bind
  • CANNOT be prevented by increasing substrate concentration (competitive can)

  A) uncompetitive

  
• mixed inhibition

  • The inhibitor binds to separate site on either the ENZYME or ES COMPLEX
  • 2 ways of getting to ESI (1 step and 2 step)

  A) ESI

  
• enzyme inhibitor examples (pharmacy)

  • aspirin: IRREVERSIBLE inhibition of COX
  • statins: REVERSIBLE, competitive inhibition of HMG-CoA reductase
• competitive inhibition - lineweaver-burk

  • Km increases as [Inhibitor] increases
  • same y-int = Vmax doesn't change

  
• Uncompetitive inhibition - lineweaver-burk

  • y-int and x-int increase as [Inhibitor] increases
  • this means that Vmax is decreases as [Inhibitor] increases
  • inhibitor binds to ES and removes space for substrate to bind to enzyme, thus lower Vmax

  
• Mixed inhibition - linewaeaver-burk

  • Vmax decreases as [Inhibitor] increases (y-int increases)
  • Km increases as [Inhibitor] increases (x-int increases)

  
• regulatory enzymes

  • to control the rate of metabolic pathways or reactions
  • eg. if need more or less of a product, these enzymes regulates this rate
• regulatory enzymes --> allosteric enzymes

  • type or regulatory enzyme
  • are quaternary structures (aka. multi-domain)
  • involves reversible and non-covalent binding of allosteric modulators/effectors onto a SEPARATE SITE of the enzyme (not active site)
  • when binded, conformational change occurs which affects substrate binding (bad and good)

  A) allosteric

  
• positive allosteric enzyme example

  • C=catalytic domain
  • R=regulatory domain
  • 1)substrate can't fit initially
  • 2) positive allosteric modulator binds to regulatory domain
  • 3) conformational change occurs ALLOWING substrate to bind

  
• allosteric enzyme KINETICS

  • if positive modulator is involved
  • low activity T-state: enzyme when it has not binded to substrate
  • high activity R-state: enzyme when there is lots of substrate (undergoes positive conformational change to accept substrate better)