enzymes

• what are the general features of enzymes?

  • usually proteins (some RNA molecules can act as enzymes - called ribozymes)
  • tend to be fairly large molecules
  • active site - small part that's used to catalyse
  • some have allosteric sites - ligands bind to these to control rate and/or specificity
  • chiral - shows stereospecificity
  • some need cofactors (coenzymes) - metal ions or organics like NADH
• specificity varies depending on the enzymes function
• specificity to the sequence - e.g. in Trypsin, R1 wants a positive charged side chain and doesn't care whats on R2

  
• an enzyme reaction is a chemical reaction - where the laws of thermodynamics still apply:

  
• The tendency for a reaction to reach equilibrium with enzymes mis driven by the Gibbs free energy:

  • if K>1 it implies the products is favoured so equilibrium lies to the right - delta G is negative
  • if K=1 implies no preference for the reaction to proceed either way - delta G is zero (there's no net gain)
  • if K<1 implies reactants favoured - delta G is positive (non-spontaneous in the forward direction

  
• enzymes don't alter the equilibrium constant or the equilibrium position, it allows equilibrium to reach faster by lowering the activation energy - rate faster and same amount of product is formed
• enzymes catalyses the forward and backward reaction by the same amount - it speeds up the rate of equilibrium by increasing both kf and kb equally

  so if kf(enzyme) is catalysed by 10, the reverse (kb) will be catalysed by 10 too.

  
• Whats the transition state?

  Enzymes helps bring the substrate molecules into the orientation to form a transition state - without the enzyme the transition states forms slower. The transition state is normally too unstable (high energy state) so short lived (transient) so it can go back to a substrate or form the product - the delta G of the reaction decides which is more favourable

  
• why is the reaction faster with an enzyme?

  The enzyme bound transition state is more stable (lower energy). For the catalysed reaction, the Gibbs free energy of activation is less and so lowers the energy needed for the substrate to reach the transition state.

  
• how does the delta G decide whether the transition state reverts back to substrate or proceeds forward to form the products?

  • if ΔG is negative - the transition state is more likely to proceed forward
  • if ΔG is positive - reaction is non-spontaneous in the forward direction and so more likely to revert back
  • if ΔG is 0 - transition state is equally likely to proceed forward or revert back
• if enzyme stabilises the transition state, it lowers the activation energy making it more favourable for the reaction to proceed forward
• describe the induced fit model?

  • substrate will bind and the enzyme will adjust the shape to induce the fit into the substrate and the fit will cause tension so it reaches the transition state better/easier for breakdown
  • active site is moulded after binding the substrate so that it fits it more precisely
• what was the earliest model for enzyme/substrate binidng?

  Lock and Key model
• what are the special features of an enzyme reaction?

  • enzymes can work in series where one enzyme forms a product which is a substrate of the next enzyme and the next enzyme. - when they are connected like this you can get product inhibition
  • this is important in the regulation of the activity of the first enzyme
  • denaturation increases with time
• what affects enzyme rates?

  Enzymes rates are affected by [E], [S], pH, temperature, additives, ionic strength, organic solvents
• whats the equation for the rate of reaction?

  V = change in concentration ÷ change in time

  • this can be written as a derivative of concentration with respect to time as $d[x]/dt$
• whats zero order?

  Zero order refers to a reaction where the rate of the reaction is independent of the concentration of the reactant.

  A goes to products - the rate is dependant on the rate constant

  Doubling the concentration of the reaction doesn't double the reaction rate - the rate remains constant as long as there is an excess of the reactant

  
• whats first order?

  Rate of the reaction is directly proportional to the concentration of the reactant raised to power of 1.

  Reaction dependant on how much you have of A

  Enzymes often display this order often

  
• whats 2nd order?

  Rate varies with concentrations of two reactants - proportional to the product of the concentrations of thee two reactants

  
• what is sudo first order?

  when one of A or B is so high in concentration that rate remains constant
• Rate varies with time:

  • there's a mix of orders during the reaction - 1st to start with and then approaches zero order towards the end
  • graph curves as concentration of substrate decreases - rate slows down
  • the rate (dP/dt) is a tangent to the curve

  
• Initial linear region gives initial rate V0 = dP/dt

  • more substrate at start = the first order is higher and so rate is higher

  
• what is an ES (Michaelis complex)?

  ES - not a transition state but a physical complex that forms and breaks down to enzyme and product. The complex can change to product or dissociate back

  In the initial part almost all the enzyme is in the form of ES (because there’s excess substrate) and product concentration is negligible so k-2 can be ignored

  
• when k-2 is ignored equation can be written out like:

  where kcat = k catalysis

  equation now is a first order expression

  
• what does the Michaelis-Menten equation describe?

  the hyperbolic dependance - provides a relationship between the reaction rate and the substrate concentration

  A) maximal velocity

  B) Michaelis constant

  C) moles/sec

  D) M

  
• what is Km?

  the concentration of the substrate where the Vmax is half - 50% saturation

  Its a measure of the enzyme's affinity for its substrate - a lower Km = higher substrate affinity
• what is Vmax?

  Maximum velocity
• what does the Michaelis-Menten equation help you work out?

  The kinetic parameters - Km, Vmax and kcat

  • these can tell you how efficient an enzyme is + how good your inhibititor might be working
• what assumptions are made when using the Michaelis-Menten equation?

  • production of product remains linear with time - working under the initial velocity conditions
  • the concentration of substrate exceeds the concentration of enzyme
  • a single enzyme forms the product
  • there is negligible product without enzyme
  • no cooperativity, allosteric modulation or any inhibition - one binding site
• what is the Michaelis-Menten equation?

  what are these labels...

  A) V0

  B) Vmax

  C) Km

  
• what does it mean when [S0]<<Km in the Michaelis-Menten equation?

  [S0] is much smaller than Km so (Km + [S0]) is almost the same as Km -

  so this is where the linear part of the curve is.

  A) Km

  B) directly

  C) first

  
• what does it mean when [S0]>>Km?

  Km is so small that (Km + [S0]) is almost the same as [S0] so:

  A) zero

  B) independant

  
• what does it mean when [S0] = Km?

  this here:

  A) Km

  B) half

  
• what orders is enzyme a mixture of?

  first and zero, depending what concentration the substrate is being used at
• what does the Michaelis-Menten equation explain?

  the hyperbolic dependance on [S0] and linear dependence on [E0]
• • when plotting initial velocity with enzyme concentration - there'll be a linear curve if there's a lot of substrate
  • at constant [S0], initial velocity is always proportional to initial enzyme concentration because all the other terms in the equation are constants

  
• why is an enzyme assay best done at S0>>Km?

  because the equation can be simplified to V0 = Vmax (zero order) - which allows the assay to operate in the linear portion of the Michaelis-Menten curve. Provides a more straightforward determination of enzyme activity.

  In linear range, its easier to detect the effects of inhibitors on enzyme activity
• what does it mean if k-1>>Kcat?

  Km is a dissociation constant for ES complex so under these conditions Km can reflect the affinity of enzyme for substrate i.e. high Km means weak binding and low affinity

  
• is a lower Km or higher Km better?

  the lower the Km the better the enzyme - it means it needs less substrate to saturate 50% of its sights
• what is Kcat?

  the turnover number - its a measure of enzyme activity as it gives an idea of its efficiency

  Its the number of substrate molecules processed per second per active site when enzyme is fully saturated with substrate (Vmax) - so its related to VmaxKcat is the breakdown of ES to product