biochem - enzymes

why do we use enzymes?

1. alter rate of reaction: concentration, temp, SA, catalyst

2. catalyst can later reaction rates → lowering activation energy barrier, pathway, stabilization TS

3. key feature about catalyst is not consumed by reaction, alter fwd and rev rxn rate, increases rate of approach to equilib

• operate under mild conditions and respond rapidly to changes

why do enzymes require non-protein species for function?

• proteins cant always do their job with the functional groups

• cofactors: simple metal ions

  • coenzymes: derived from vitamins, tightly bound to enzyme ; some act as cosubstrates

what is michaelis-menten rules?

• S → P, assuming unimolecular

• where the observed fwd rate = true foward rate - true reverse rate = k1S- k1P

  • where k1= fwd rate constant, k-1= rev rate constant

how does graphing on a Vo vs S plot look like? factors?

• influence of S on initial velocity

• hyperbolic plot, first order relationship

  • prepare initial velocity vs S plot

  • use that value to find Vmax: the maximum initial velocity

    • saturation kinetics: top asymptote of the rectangular hyperbola; alternate grpahing protocols can give a better estimate of Vmax

  • use Vmax to find Km which is the binding affinity, where S must meet ½ of it

what are the assumptions of the derivation of MM equation?

1. assume S»E thus the value of S remains essential constant during the inital velocity data collection, little product

2. ES remains essential constant once a steady state is established

3. assume we know that the rate-limiting step is ES → E +P

4. only forms of enzyme are E and ES thus E total = E + ES

what is the physical significance of km?

• sometimes an index of affinity of binding between E and S

• lower the km stronger the affinty

• used to compare relative affinity for different potential substrates for the same enzyme

what is the significance of Vmax?

• values vary widely and depend on amount of enxyme used in export

• better value to use if Vmax/E total or kcat the turnover number

what is kcat or the turnover number?

• number of substrate molecules converted to product on a single enzyme molecule active site per second when the enzyme is saturated with substrate

*what is the best way to do comparisons of different enzymes with respect to efficienty of their active sites?

• use the ratio of kcat/km → specificty constant: index of catalytic efficiency under celluar conditions

  • kcat: considers efficiency

  • km: considers the strenght of ES binding interaction

  • upper limit: diffusion limited rxns

    • but multienzyme complexes can negate this

what is the function of the sequential bibi reaction?

• all substrates must combine with the enzyme before a reaction can occur and products can be release

• species transferred from one substrate is directly passed to the other

  • A(p-x) pass x to B → P and Q (b-x)

what are the subclassifications of sequential bibi rxns?

• ordered: binding 1st substrate is required for the enzyme to be able to bind to the second

• random: no preference in order

what is the function of the ping pong bibi rxn?

• one product is released before all substrates associate with the enzyme

  • A(p-x) → displaced → P + F (e-x)

• then group X is displaced from the enzyme by the 2nd substrate to yeild the 2nd product thus regenerating the enzyme to orginal form

• substrate A and B do not encounter each other

how does temperature alter enzyme activity?

• low temp: free energy effects

• high temp: weak interactions broken / weakend

how does pH alter enzyme activity?

• charge state of AA needed of substrate binding

• charge state of AA at active site

• charge state of substrates

• protein structure (extreme tho)

what is a competitive inhibitor (rv)?

1. I binds reversibly to E

2. S and I can not be both bound to E

3. EI complex is inactive

4. Ki- dissociate constant of EI complex

5. Etotal= E + ES + EI

• km: inc, vmax: no change

  • since there is an inhibitor competeing with the substrate to bind to the enzyme, we most increase the concentration of the substrate to outcompete it therefore the higher concentration increases the need to reach that Vmax increasing km, whereas the amount of binding does not change so Vmax does not change

what is a uncompetitve inhibitor (rv)?

1. I binds reversibly to the ES complex

2. ESI complex is inactive

3. Ki= dissociation of ESI

4. Etotal = E + ES + ESI

• km: dec, vmax: dec

  • here the inhibitor locks the ES/ESI complex into place increasing the binding affinity, lowering the km.Vmax decreases because the inhibitor locks the ES complex inabling product formation

what is a mixed inhibitor (rv)?

1. I binds reversibly to ES or to E

2. ESI and EI complexes are inactive

3. dissociation constates, 2 ki= EI, ki’= ESI

4. Etotal = E + ES + ESI + EI

• km: inc, vmax dec

  • here the inhibitor locks the ES/ESI complex into place increasing the binding affinity, lowering the km.Vmax decreases because the inhibitor locks the ES complex inabling product formation

what is a noncompetitve inhibitor (rv)?

• where ki=ki’

  • km: no change, vmax: dec

    • this is because the inhibition occurs at the allosteric site not the ES complex therefore not interfering with ES, Vmax decasues bc the allosteric site can turn off/on function of the ES complex limiting production

what is dialysis?

1. mix inhibitor with enzyme to allow to react fully

2. place into dialysis add stir bar

3. remove sample and test for activity

4. if reversible, there should he higher activity if not no change

what is the difference between irreversible and reversible inhibition?

reversible: association is temporary

irreversible: bind permanently to the enzyme

what are ways to experimentally determine if an inhibitor is reversible or not?

1. time course

   1. reversable: almost instant binding

   2. irreversible: time dependent, decrease in activity as EI forms

2. dialysis

   1. reversible: dialysis should decrease, due to the extent of inhibition and influences equilibrium

   2. irreversible: EI concentration would not decrease/move